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Information on EC 2.7.7.7 - DNA-directed DNA polymerase and Organism(s) Saccharolobus solfataricus and UniProt Accession P96022
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2.7.7.7 DNA-directed DNA polymeraseIUBMB Comments
Catalyses DNA-template-directed extension of the 3'- end of a DNA strand by one nucleotide at a time. Cannot initiate a chain de novo. Requires a primer, which may be DNA or RNA. See also EC 2.7.7.49 RNA-directed DNA polymerase.
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Word Map
- 2.7.7.7
- strand
-
single-stranded
-
fidelity
- bacteriophage
-
holoenzyme
- mismatch
- pcna
- fork
- polymerization
- aphidicolin
-
proofreading
- duplex
-
calf
- thymus
- endonuclease
- helicase
-
clamp
-
misincorporation
-
stall
- 5'-triphosphate
- dntps
-
template-primer
-
abasic
-
sliding
- thymine
- mispairs
-
anneal
-
uv-induced
-
error-free
- xeroderma
- pigmentosum
-
okazaki
-
cyclobutane
-
hypermutation
-
exonucleolytic
- glycosylase
-
deoxynucleotide
- ssdna
-
undamaged
- deoxyribonucleoside
- replisome
-
unwind
-
transversions
- aquaticus
-
watson-crick
-
myeloblastosis
- transcriptases
- dtmp
-
slippage
- synthesis
-
8-oxog
- drug development
- molecular biology
- diagnostics
- medicine
- analysis
- biotechnology
The taxonomic range for the selected organisms is: Saccharolobus solfataricus
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
dna polymerase alpha, dna polymerase beta, dna polymerase iii, pol beta, klenow fragment, dna polymerase delta, taq dna polymerase, pol delta, pol alpha, dna polymerase gamma, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Dbh polymerase
DNA polymerase
DNA polymerase B1
DNA polymerase Dpo4
DNA polymerase III
DNA polymerase IV
DNA-dependent DNA polymerase
Dpo1
Dpo4
SSO2448
additional information
DNA polymerase Dpo4
286134
-
DNA polymerase IV
2070
-
DNA polymerase IV
286134
-
Dpo4
2070
-
Dpo4
286134
-
690931, 690974, 695225, 704514, 718837, 719283, 719650, 719817, 719836, 719841, 720575, 720577, 721665, 724266, 724354, 724363, 724387, 724705, 724739, 724740, 724744, 724745, 724746, 724760, 724845, 725350, 725361, 725374, 725377, 725385, 725390, 725439, 725702, 725713, 725996, 726028, 726077, 726358, 729272
additional information
2070
the enzyme belongs to the DNA polymerase Y-family
additional information
2070
the enzyme belongs to the Y-family polymerases
additional information
2070
the enzyme belongs to the Y-family polymerases and is a member of the RAD30A subfamily
additional information
2070
the enzyme is a Y-family DNA polymerase
additional information
286134
the enzyme belongs to the Y-family polymerases
additional information
286134
the enzyme belongs to the Y-family polymerases
additional information
286134
the enzyme belongs to the Y-family polymerases
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
mechanism
-
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
reaction mechanism
-
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
modelling and simulation of the reaction mechanism, detailed overview
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
nucleotidyl-transfer reaction mechanism, intermediate structures and energy profiles, overview
-
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
reaction and kinetic mechanism, overview
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (DNA-directed)
Catalyses DNA-template-directed extension of the 3'- end of a DNA strand by one nucleotide at a time. Cannot initiate a chain de novo. Requires a primer, which may be DNA or RNA. See also EC 2.7.7.49 RNA-directed DNA polymerase.
CAS REGISTRY NUMBER
COMMENTARY
9012-90-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate + DNAn
diphosphate + ?
-
-
-
?
2-thio-dCTP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
5-methyl-dCTP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
7-deaza-2'-deoxyadenosine 5'-triphosphate + DNAn
diphosphate + ?
-
-
-
?
8-bromo-dATP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
8-oxo-dATP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
dATP + DNAn
diphosphate + DNAn+1
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
dCTP + DNAn
diphosphate + DNAn+1
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
deoxynucleoside triphosphate + DNAn
?
the enzyme can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene. An anti glycosidic torsion with C1'-exo deoxyribose conformation allows N-(deoxyguanosin-8-yl)-2-acetylaminofluorene to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting
-
-
?
dGTP + DNAn
diphosphate + DNAn+1
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
DNA 21/41-mer + dTTP
? + diphosphate
kinetic mechanism for DNA polymerization is proposed, the enzyme utilizes an induced-fit mechanism to select correct incoming nucleotides
-
-
?
dPTP + DNAn
?
i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-beta-D-2'-deoxyribofuranosid 5'-triphosphate. dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine
-
-
?
dTTP + DNAn
diphosphate + DNAn+1
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
N1-methyl-2'-deoxyadenosine 5'-triphosphate + DNAn
diphosphate + ?
-
-
-
?
South-methanocarba-dATP + DNAn
?
the role of sugar geometry during nucleotide selection is probed by the enzyme from Sulfolobus solfataricus using fixed conformation nucleotide analogues. The enzyme relatively tolerant to the substrate conformation: North-methanocarba-dATP that locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker or South-methanocarba-dATP that locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
in the absence of additional cofactor the enzyme is an essentially distributive enzyme that only extends primers by 1-2 nt per binding event. At high enzyme to primer/template ratios, dissociation and rebinding of the enzyme to the primer/template is robust and can lead to the synthesis of polynucleotide chains of several hundred nucleotides in length
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
ribonucleotide discrimination by the DinB homolog (Dbh) DNA polymerase is as stringent as in other polymerases. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme is nonprocessive and can bypass an abasic site
-
-
?
dATP + DNAn
?
-
Dbh is a distributive enzyme showing a low DNA and nucleotide binding affinity along with a slow polymerization rate. DNA binding occurs in a single step, diffusion-controlled manner. The rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. An induced fit mechanism to select and incorporate nucleotides during DNA polymerization can not be detected for the enzyme
-
-
?
dATP + DNAn
?
in addition to the correct insertion of dATP opposite the lesion, Dpo4 misincorporates dATP, dGTP, and TTP in an oligonucleotide containing a site-specific N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion. dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion is only 1.4fold lower than insertion opposite an unmodified deoxyguanosine
-
-
?
dCTP + DNAn
?
-
Dbh is a distributive enzyme showing a low DNA and nucleotide binding affinity along with a slow polymerization rate. DNA binding occurs in a single step, diffusion-controlled manner. The rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. An induced fit mechanism to select and incorporate nucleotides during DNA polymerization can not be detected for the enzyme
-
-
?
dCTP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-methyl-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
dCTP + DNAn
?
dTTP incorporation is the most preferred addition opposite the N6dA-(OH)2butyl-GSH adduct, N6dA-butanetriol adduct, or unmodified dA
-
-
?
dCTP + DNAn
?
in addition to the correct insertion of dCTP opposite the lesion, Dpo4 misincorporates dATP, dGTP, and TTP in an oligonucleotide containing a site-specific N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion. dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion is only 1.4fold lower than insertion opposite an unmodified deoxyguanosine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
exonuclease 3'--5' activity
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3–4 bases upstream of this lesion (read-ahead function). Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Sso DNA pol B1 physically interacts with DNA pol Y1. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617)
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
with template guanine and Watson-Crick paired dCTP as the nascent base pair. Water-mediated and substrate-assisted mechanism: the initial proton transfer to the R-phosphate of the substrate via a bridging crystal water molecule is the rate-limiting step, the nucleotidyl-transfer step is associative with a metastable pentacovalent phosphorane intermediate, and the diphosphate leaving is facilitated by a highly coordinated proton relay mechanism through mediation of water which neutralizes the evolving negative charge. The conserved carboxylates, which retain their liganding to the two Mg2+ ions during the reaction process, are found to be essential in stabilizing transition states. This water-mediated and substrate-assisted mechanism takes specific advantage of the unique structural features of this low-fidelity lesion-bypass Y-family polymerase, which has a more spacious and solvent-exposed active site than replicative and repair polymerases
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine, a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene. The diol epoxide (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene reacts largely, but not exclusively, with the exocyclic amino group of guanine to produce the major 10S (+) trans-anti-BP-N2-dG adduct, that is bypassed by Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
mechanism of purine-purine mispair formation, substrate specificity and binding structure, the kpol/Kd dNTP values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine are decreased over 10fold with respect to those of the unmodified nucleotides during formation of purine-purine mispairs. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine is decreased 5fold. Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations, overview
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG adduct in DNA polymerase IV, 5'-slippage mechanism: the dATP can be inserted opposite the T on the 5' side of the adduct G1*, in which the unadducted G2, rather than G1*, is skipped, to produce a -1 deletion, molecular modeling and dynamics simulations, overview
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
substrate is a 44-mer DNA template containing a site-specific cisplatin-d(GpG) adduct, Dpo4 is able to bypass a single, site-specifically placed cisplatin-d(GpG) adduct, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases is decreased by 72 and 860fold, respectively, enzyme fidelity, overview
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis, correct insertion of dCTP opposite 8-oxoguanine and quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization, overview
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine, a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, Dpo4 bypasses 8-oxoG accurately
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
approximately 2/3 of the errors made by the enzyme are single-base substitutions, of which 58% are C->T transition. Frameshift mutations, mostly resulting from single-base deletions, account for 19% of the total errors. An exonuclease-deficient mutant of Sso pol B1 is three times as mutagenic as the wild-type enzyme, suggesting that the intrinsic proofreading function contributed only modestly to the fidelity of the enzyme
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
bypass of apurinic/apyrimidinic sites lacking A or G is nearly 100% mutagenic. The majority (70–80%) of bypass events are insertion of dAMP opposite the apurinic/apyrimidinic site
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
DNA polymerase IV (Dpo4) shows 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. Extension beyond an 8-oxoG:C pair is similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. dCTP insertion opposite 8-oxoG was lower than for opposite guanine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
modeling and molecular dynamics simulations for 2'-deoxy-8-[(1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridin-2-yl)amino]guanosine suggest that the adduct would increase the infidelity of Dpo4 and hinder translocation by the enzyme
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
products of replication of polycyclic aromatic hydrocarbon-modified DNA by the translesion DNA polymerase Dpo4 are complex
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
propenal and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-beta-D-erythro-pentofuranosyl) pyrimido[1,2-alpha]purin-10(3H)-one (M1dG). When paired opposite cytosine in duplex DNA at physiological pH, M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG. To improve the understanding of the basis for M1dG-induced mutagenesis, the mechanism of translesion DNA synthesis opposite M1dG by the model Y-family polymerase Dpo4 is studied at a molecular level using kinetic and structural approaches. The enzyme can bypass the exocyclic M1dG adduct in largely an error-prone fashion
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
replication bypass studies in vitro reveal that the polymerase inserts dNTPs opposite the (6S,8R,11S)-trans-4-hydroxynonenal-1,N2-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base is dCyt, the polymerase inserts primarily dGTP. If the template 5'-neighbor base is dThy, the polymerase inserts primarily dATP
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme bypasses aflatoxin B1-N7-dG in an error-free manner but conducts error-prone replication past the aflatoxin B1-formamidopyrimidine adduct, including misinsertion of dATP
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme shows a limited decrease in catalytic efficiency (kcat/Km) for insertion of dCTP opposite a series of N2-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity is maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increases slightly going from the N2-NaphG to the N2-AnthG substrate, at the cost of fidelity. A set of oligonucleotides differing only in their N2-substitution at a single G site is used in this study with Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the pre-steady-state kinetic methods is used to determine the base substitution fidelity and mismatch extension fidelity of PolB1
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the rate-constant defining Dpo4-catalyzed incorporation of dCTP is about 6-fold slower for incorporation opposite O6-MeG relative to G. The basis for the decreased rate is revealed by the crystal structure to be formation of a wobble base pairing between O6-MeG and C
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
translesion synthesis of the 7-(2-oxoheptyl)-1,N2-etheno-2'-deoxyguanosine lesion by the enzyme in 5'-TXG-3' and 5'-CXG-3' local sequence contexts is examined and compared to 1,N2-etheno-2'-deoxyguanosine lesions
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme can efficiently incorporate nucleotides opposite 8-oxoG and extend from an 8-oxoG:C base pair with a mechanism similar to that observed for the replication of undamaged DNA
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. B-family DNA polymerases from hyperthermophilic archaea recognize the presence of uracil in DNA and stall DNA synthesis. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency is substantially enhanced by the initiation of DNA synthesis. The generation of ds DNA is significantly inhibited, however, by the presence of template uracil. Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution. Single molecules of Pol B1 bind to template uracil and stall DNA synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme bypasses DNA adducts pyrrolo-deoxycytosine, dP, N6-furfuryl-deoxyadenosine, and 1,N6-ethenodeoxyadenosine in a process known as translesion synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis 2-OH-dATP is predominantly incorporated opposite guanine and thymine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis. 2-OH-dATP is predominantly incorporated opposite guanine and thymine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
an abasic lesion causes Dpo4 to switch from a normal to a very mutagenic mode of replication. Incorporation upstream of the abasic lesion is replicated error-free. Once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37°C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. The percentage of overall frameshift mutations increases from 71% (37°C) to 87% (75°C)
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
an abasic lesion causes the enzyme to switch from a normal to a very mutagenic mode of replication
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
ATP binding to replication factor C is sufficient for loading the heterotrimeric PCNA123 [proliferating cell nuclear antigen (PCNA)] clamp onto DNA that includes a rate-limiting conformational rearrangement of the complex. ATP hydrolysis is required for favorable recruitment and interactions with the replication polymerase (PolB1) that most likely include clamp closing and dissociation of replication factor C. Surprisingly, the assembled holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. PolB1 repeatedly disengages from the DNA template, leaving PCNA123 behind. Interactions with a C-terminal PCNA-interacting peptide (PIP) motif on PolB1 specifically with PCNA2 are required for holoenzyme formation and continuous re-recruitment during synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
bifunctional enzyme EC 2.7.7.7/EC 3.1.11.2. The polymerization and the 3'-5' exonuclease activity of a family B DNA polymerase can be ascribed to physically distinct modules of the enzyme molecule
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
Dbh polymerase is much less accurate than the classical polymerases, but it shows a remarkable tendency to skip over a template pyrimidine positioned immediately 3' to a G residue, generating a single-base deletion. The rate of incorporation of dCTP opposite a template G is about 10fold faster than for the other three dNTPs opposite their complementary partners
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of proliferating cell nuclear antigen (039p or 048p) and replication factor CRFC. The length of the synthesized DNA product reaches at least 200 nucleotides
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
DNA polymerase Dpo4 can replicate past a variety of DNA lesions. When replicating undamaged DNA, the enzyme is prone to make base pair substitutions
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
DNA polymerase pol Y1 exclusively incorporates 8-OH-GTP opposite adenine. DNA polymerase pol Y1 incorporates 2-OH-dATP predominantly opposite guanine and thymine. DNA polymerase pol B1 incorporates 8-OH-GTP opposite adenine and cytosine. DNA polymerase pol B1 incorporates 2-OH-dATP opposite thymine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
Dpo4 in most cases selects the correctly paired partner for each benzo-expanded DNA base, but with efficiency lowered by the enlarged pair size
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
dTTP incorporation is the most preferred addition opposite the N6dA-(OH)2butyl-GSH adduct, N6dA-butanetriol adduct, or unmodified dA
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
even at 60°C, excessive amounts of Dpo4 are needed to carry out minimal bypass of the cyclobutane pyrimidine dimers
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
exclusively incorporates 8-OH-GTP opposite adenine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
GTP incorporation by the wild-type enzyme is about 1000fold slower than dGTP incorporation. The rate of GTP incorporation by the mutant enzyme F12A Dbh is 2-3fold slower than incorporation of dGTP. The enzyme makes single-base deletion errors at high frequency in particular sequence contexts
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
in addition to the correct insertion of dCTP opposite the lesion, Dpo4 misincorporates dATP, dGTP, and TTP in an oligonucleotide containing a site-specific N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
incorporated of 2-amino-3-methylimidazo[4,5-f]quinoline C8- and N2-dGuo adducts into the G1- and G3-positions of the NarI recognition sequence (5'-G1G2CG3CC-3'), which is a hotspot for arylamine modification. Replication of the C8-adduct at the G3-position results in two-base deletion, whereas error-free bypass and extension is observed at the G1-position. The N2-adduct is bypassed and extended when positioned at the G1-position, and the error-free product is observed. The N2-adduct at the G3-position is more blocking and is bypassed and extended only by Dpo4 to produce an errorfree product. The replication of the 2-amino-3-methylimidazo[4,5-f]quinoline-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
low fidelity. When copying undamaged DNA, Dpo4 is highly inaccurate for essentially all types of single base substitutions and deletions in a large number of different sequence contexts
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
mechanism of template-independent nucleotide incorporation. Based on the efficiency ratios, Dpo4 selects nucleotides for blunt-end addition in the order of decreasing efficiency: dATP, dTTP, dCTP, dGTP, with dATP favored by five to 50fold over the other nucleotides. The first bluntend dATP incorporation is 80fold more efficient than the second, and among natural deoxynucleotides, dATP is the preferred substrate due to its stronger intrahelical base-stacking ability
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
proliferating cell nuclear antigen facilitates DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein is most processive with DNA polymerase Dpo1 in comparison to DNA polymerase Dpo3 and Dpo4. DNA lesion bypass DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein is most effective with DNA polymerase Dpo4 in comparison to DNA polymerase Dpo1 and Dpo3. Both Dpo2 and Dpo3, but not Dpo1, bypass hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypass uracil and cis-syn cyclobutane thymine dimer, respectively. DNA polymerase Dpo2 and Dpo3 possess very low DNA polymerase and 3' to 5' exonuclease activities in vitro compared with Dpo1 and Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
relative to undamaged DNA the enzyme generates far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed N-(deoxyguanosin-8-yl)-1-aminopyrene. Opposite N-(deoxyguanosin-8-yl)-1-aminopyrened and at an immediate downstream template position, the most frequent base substitutions are dA
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
removal of the 2-amino group from the template dG (i.e. deoxyinosine) has little impact on the catalytic efficiency of either polymerase, although the misincorporation frequency is increased by an order of magnitude. Deoxyxanthosine is highly miscoding with both polymerases, and incorporation of several bases is observed. The addition of bromine or oxygen at C2 lowers the Tm further, strongly inhibits and increases the frequency of misincorporation
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
ring-opening of the 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one adduct promotes error-free bypass by the Sulfolobus solfataricus DNA polymerase Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the binding of a correct nucleotide induces a fast and surprising DNA translocation event. All four domains of the polymerase rapidly move in a synchronized manner before and after the polymerization reaction. Repositioning of active site residues is the rate-limiting step during correct nucleotide incorporation. The motions of the polymerase and the polymerase bound DNA substrate are tightly coupled to catalysis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the conformational dynamics of the Y-family DNA polymerase Dpo4 on DNA is characterized in real time using single-molecule Förster resonance energy transfers (mFRET). Two different binary complexes consistent with DNA translocation in the polymerase active site
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme binds to DNA in at least three distinct conformations. The relative frequency of each conformation can be modulated by both the identity of the primer 3' terminus and the presence of an incoming dNTP
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme bypasses Pt-GG DNA (1,2-intrastrand covalent linkage, cis-Pt-1,2-d(GpG)). This is a dynamic process, in which the lesion is converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme does not efficiently insert nucleotides opposite to the (6S,8R,11S)-1,N2-deoxyguanosine adduct, consistent with the low levels of Gua->Thy mutations. However it extends past the (6S,8R,11S)-1,N2-deoxyguanosine:dCyd pair. A series of ternary (Dpo4-DNA-dNTP) structures with (6S,8R,11S)-1,N2-deoxyguanosine-adducted templates suggest that during replication, the ring-closed (6S,8R,11S)-1,N2-deoxyguanosine lesion at the active site hinders incorporation of dNTPs opposite the lesion, whereas the ring-opened form of the lesion in the (6S,8R,11S)-1,N2-deoxyguanosine:dCyd pair allows for extension to full-length product
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme incorporates dTTP in a 61 mer template containing pyrrolo-deoxycytosine, dP, N6-furfuryl-deoxyadenosine, and 1,N6-ethenodeoxyadenosine (translesion synthesis)
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme is able to bypass N-(deoxyguanosin-8-yl)-1-aminopyrene, but pauses strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decrease significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter inding affinity of damaged DNA to Dpo4 DNA polymerase promotes catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme is inefficient at extending mispairs opposite a template G or T, which include, a G*T mispair expected to conform closely to Watson-Crick geometry. It is hindered in extending a G*T mismatch by a reverse wobble
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allows for template-directed synthesis at temperatures more than 30°C above the melting temperature of naked DNA. Dpo1 elongates single stranded DNA in template-dependent and template-independent manners. Initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. Dpo1 also displays a competing terminal deoxynucleotide transferase activity unlike any other B-family DNA polymerase
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme shows little decreases opposite N2-MeG, 13fold decrease opposite N2-BzG but 260-370fold decreases opposite O6-MeG, O6-BzG, and the abasic site site as compared to G. Dpo4 favored correct C insertion opposite opposite N2-MeG, opposite O6-MeG, opposite an abasic site site and oppositeN2-BzG. DNA polymerase Vent (exo-) from Thermococcus litoralis is as or more efficient as polymerase Dpo4 from Sulfolobus solfataricus in synthesis opposite O6-MeG and AP lesions, whereas DNA polymerase Dpo4 from Sulfolobus solfataricus is much or more efficient opposite N2-alkylGs than DNA polymerase Vent (exo-) from Thermococcus litoralis, irrespective of DNA-binding affinity. DNA polymerase Dpo4 strongly favors minor-groove N2-alkylG lesions over major-groove or noninstructive lesions
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the fidelity of Dpo4 is in the range of 0.001-0.0001. The ground-state binding affinity of correct nucleotides is 10-50-fold weaker than those of replicative DNA polymerases. The affinity of incorrect nucleotides for Dpo4 is about 2-10-fold weaker than that of correct nucleotides. The mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The mismatch incorporation rates, regardless of the 5'-template base, are about 2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by the enzyme, with hydrogen bonding capacity being a major influence. Modifications at the C2-position of dCTP increases the selectivity for incorporation opposite O6-methylguanine without a significant loss of efficiency
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the initial enzyme/DNA/dNTP complex undergoes a rapid (18/s), reversible (21/s) conformational change, followed by relatively rapid phosphodiester bond formation (11/s) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2/s) and then rapid DNA release, yielding an overall steady-state kcat of less than 1/s
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the molecular dynamics simulations suggest that mismatched nucleotide insertion opposite 10S-(+)-trans-anti-[benzo[a]pyrene]-N2-dG is increased at 55°C compared with 37°C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. With the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite 10S-(+)-trans-anti-[benzo[a]pyrene]-N2-dG by Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the rate of insertion of dNTPs opposite and extension past both O2-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine and O2-methylthymidine is measured. The size of the alkyl chain only marginally affects the reactivity and the specificity of adduct bypass is very low. Dpo4 catalyzes the incorporation opposite and extension past the adducts approximately 1000fold more slowly than undamaged DNA. dA is the preferred base pair partner for O2-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine and dT is the preferred base pair partner for O2-methylthymidine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the rate of mismatched nucleotide incorporation is greater than the rate of correct dC insertion at 55 °C, whereas at 37 °C there is little selectivity
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
when insertion is opposite an unmodified G, insertion of dATP or dGTP is 1000 less efficient than dCTP. For insertion opposite 8-oxoG, the order of decreasing efficiency is dCTP, dATP, dGTP, with an order of magnitude or more difference in catalytic efficiency (kcat/Km) in each pair of comparisons. The insertion of dCTP opposite G and 8-oxoG shows similar catalytic efficiency, even with differences in the trends for kcat and Km. 90fold higher incorporation efficiency of dCTP compared to dATP opposite 8-oxoG and 4fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. The 8-oxoG:C pair shows classic Watson-Crick geometry; the 8-oxoG:A pair is in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5'C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshiftevent observed in the catalytic assays
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
while showing efficient bypass, the enzyme pauses when incorporating nucleotides directly opposite and one position downstream from an abasic lesion because of a drop of several orders of magnitude in catalytic efficiency. Biphasic kinetics for incorporation indicating that Dpo4 primarily forms a nonproductive complex with DNA that converts slowly to a productive complex. These strong pause sites are mutational hot spots with the embedded lesion even affecting the efficiency of five to six downstream incorporations
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme inefficiently bypasses (5'S)-8,5'-cyclo-2'-deoxyguanosine with dCTP preferably incorporated and dATP misincorporated. (5'S)-8,5'-cyclo-2'-Deoxyguanosine attenuates K(d,dNTP,app) and k(pol)
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme inefficiently bypasses (5'S)-8,5'-cyclo-2'-deoxyguanosine with dCTP preferably incorporated and dTTP misincorporated. The (5'S)-8,5'-cyclo-2'-Deoxyguanosine-adduct-duplex complex causes 6fold decrease in Dpo4:DNA binding affinity, and significantly reduces the concentration of the productive Dpo4:DNA:dCTP complex
-
-
?
dGTP + DNAn
?
-
Dbh is a distributive enzyme showing a low DNA and nucleotide binding affinity along with a slow polymerization rate. DNA binding occurs in a single step, diffusion-controlled manner. The rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. An induced fit mechanism to select and incorporate nucleotides during DNA polymerization can not be detected for the enzyme
-
-
?
dGTP + DNAn
?
in addition to the correct insertion of dGTP opposite the lesion, Dpo4 misincorporates dATP, dGTP, and TTP in an oligonucleotide containing a site-specific N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion. dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion is only 1.4fold lower than insertion opposite an unmodified deoxyguanosine
-
-
?
dTTP + DNAn
?
-
Dbh is a distributive enzyme showing a low DNA and nucleotide binding affinity along with a slow polymerization rate. DNA binding occurs in a single step, diffusion-controlled manner. The rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. An induced fit mechanism to select and incorporate nucleotides during DNA polymerization can not be detected for the enzyme
-
-
?
dTTP + DNAn
?
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
dTTP + DNAn
?
dTTP incorporation is the most preferred addition opposite the N6dA-(OH)2butyl-GSH adduct, N6dA-butanetriol adduct, or unmodified dA
-
-
?
dTTP + DNAn
?
in addition to the correct insertion of dTTP opposite the lesion, Dpo4 misincorporates dATP, dGTP, and TTP in an oligonucleotide containing a site-specific N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion. dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion is only 1.4fold lower than insertion opposite an unmodified deoxyguanosine
-
-
?
North-methanocarba-dATP + DNAn
?
the role of sugar geometry during nucleotide selection is probed by the enzyme from Sulfolobus solfataricus using fixed conformation nucleotide analogues. The enzyme preferentially inserts North-methanocarba-dATP that locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker compared to a South-methanocarba-dATP that locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker
-
-
?
North-methanocarba-dATP + DNAn
?
the role of sugar geometry during nucleotide selection is probed by the enzyme from Sulfolobus solfataricus using fixed conformation nucleotide analogues. The enzyme relatively tolerant to the substrate conformation: North-methanocarba-dATP that locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker or South-methanocarba-dATP that locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker
-
-
?
additional information
?
-
replication cycle of Dpo4, and induced fit and translocation mechanisms, overview
-
-
?
additional information
?
-
-
replication cycle of Dpo4, and induced fit and translocation mechanisms, overview
-
-
?
additional information
?
-
-
the widely used anticancer drug, cis-diamminedichloroplatinum(II), i.e. cisplatin, reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2))] intrastrand cross-links, DNA polymerase IV is able to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin-DNA adducts, overview
-
-
?
additional information
?
-
Dpo4 produces mismatch and frameshift mutations at benzo[a]pyrene-derived lesions, overview
-
-
?
additional information
?
-
-
Dpo4 produces mismatch and frameshift mutations at benzo[a]pyrene-derived lesions, overview
-
-
?
additional information
?
-
-
Dpo4 utilizes an induced-fit mechanism to select correct incoming nucleotides at 37°C, overview
-
-
?
additional information
?
-
-
potential structures of purine-purine base pairs, overview
-
-
?
additional information
?
-
significant preferential dATP insertion, dATP can be misincorporated opposite the benzo[a]pyrene-derived N2-dG adduct, standing-start single-nucleotide insertion assays, overview
-
-
?
additional information
?
-
-
significant preferential dATP insertion, dATP can be misincorporated opposite the benzo[a]pyrene-derived N2-dG adduct, standing-start single-nucleotide insertion assays, overview
-
-
?
additional information
?
-
-
simulation model of the solvated Dpo4/DNA/8-oxoG:dCTP complex, catalytic site structure, overview
-
-
?
additional information
?
-
Dpo3 has an active exonuclease proofreading domain, it shows intrinsic exonuclease activity
-
-
?
additional information
?
-
-
Dpo3 has an active exonuclease proofreading domain, it shows intrinsic exonuclease activity
-
-
?
additional information
?
-
-
simulations, based on crystal complexes of Dpo4 are performed, exploring possible transitions and mechanisms associated with Dpo4’s catalytic cycle. Dynamics simulations before the nucleotidyl-transfer reaction and simulations after the reaction are performed. Subtle but variable conformational rearrangements in the replication cycle of Sulfolobus solfataricus P2 DNA polymerase IV may accommodate lesion bypass
-
-
?
additional information
?
-
-
the DNA lesion bypass polymerase can bind up to eight base pairs of double-stranded DNA which is entirely in B-type. Thus, the DNA binding cleft of Dpo4 is flexible and can accommodate both A- and B-type oligodeoxyribonucleotide duplexes as well as damaged DNA
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine, a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene. The diol epoxide (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene reacts largely, but not exclusively, with the exocyclic amino group of guanine to produce the major 10S (+) trans-anti-BP-N2-dG adduct, that is bypassed by Dpo4
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. B-family DNA polymerases from hyperthermophilic archaea recognize the presence of uracil in DNA and stall DNA synthesis. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency is substantially enhanced by the initiation of DNA synthesis. The generation of ds DNA is significantly inhibited, however, by the presence of template uracil. Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution. Single molecules of Pol B1 bind to template uracil and stall DNA synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the enzyme bypasses DNA adducts pyrrolo-deoxycytosine, dP, N6-furfuryl-deoxyadenosine, and 1,N6-ethenodeoxyadenosine in a process known as translesion synthesis
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis 2-OH-dATP is predominantly incorporated opposite guanine and thymine
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis. 2-OH-dATP is predominantly incorporated opposite guanine and thymine
-
-
?
additional information
?
-
replication cycle of Dpo4, and induced fit and translocation mechanisms, overview
-
-
?
additional information
?
-
-
replication cycle of Dpo4, and induced fit and translocation mechanisms, overview
-
-
?
additional information
?
-
-
the widely used anticancer drug, cis-diamminedichloroplatinum(II), i.e. cisplatin, reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2))] intrastrand cross-links, DNA polymerase IV is able to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin-DNA adducts, overview
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
DTT
Mg2+
Mn2+
Ca2+
Ca2+ (but not Ba2+, Co2+, Cu2+, Ni2+, or Zn2+) is a cofactor for Dpo4-catalyzed polymerization with both native and 8-oxoG-containing DNA templates. Both dNTP and ddNTP are substrates of the polymerase in the presence of either Mg2+ or Ca2+. No pyrophosphorolysis occurs in the presence of Ca2+
Mg2+
cofactor for both the polymerase and pyrophosphorolysis activities
Mg2+
DNA polymerase Dpo2 and Dpo3 are both more active with Mg2+ than Mn2+. DNA polymerase Dpo1 and Dpo4 are similarly active with Mg2+ or Mn2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3-(2'-deoxy-beta-D-erythro-pentofuranosyl) pyrimido[1,2-alpha]purin-10(3H)-one
-
i.e. M1dG. When paired opposite cytosine in duplex DNA at physiological pH,M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG. To improve the understanding of the basis for M1dG-induced mutagenesis, the mechanism of translesion DNA synthesis opposite M1dG by the model Y-family polymerase Dpo4 is studied at a molecular level using kinetic and structural approaches. Steady-state and transient-state kinetic results both indicate that Dpo4 catalysis is inhibited by M1dG (260-2900-fold), with dATP being the favored insertion event for both sequences tested
cis-syn thymine dimer
Dpo4 is severely blocked by the presence of a cis-syn thymine dimer in the template
N2,N2-dimethyl guanine
the presence of N2,N2-dimethyl guanine lowers the catalytic efficiency for incorporation of dCTP of DNA polymerase Dpo4 16000fold
N2,N2-dimethylguanine
the presence of N2,N2-dimethylguanine lowers the catalytic efficiency of the DNA polymerase Dpo4 16000fold. Dpo4 inserts dNTPs almost at random during bypass of N2,N2-dimethylguanine, and much of the enzyme is kinetically trapped by an inactive ternary complex when N2,N2-dimethylguanine is present
Replication factor C
DNA polymerization by Dpo2 and Dpo3 is strongly inhibited
-
SsoCdc6-2 protein
-
inhibits both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it forms a large complex with SsoPolY and stimulates DNA-binding activity on paired primer-template DNA substrates
-
additional information
-
reverse gyrase inhibits DNA polymerase PolB1. Inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control DNA polymerase PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a mechanism of regulation of these enzymes
-
single-stranded binding protein
DNA polymerization by Dpo2 and Dpo3 is strongly inhibited
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SsoCdc6
-
Orc1/Cdc6 proteins stimulate the DNA-binding ability of SsoPolB1 and differentially regulate both its polymerase and nuclease activities
-
proliferating cell nuclear antigen
DNA synthesis by any of the DNA polymerases alone is not very processive. The addition of proliferating cell nuclear antigen slightly increases primer extension only by DNA polymerase Dpo4 but not by DNA polymerase Dpo1, Dpo2, or Dpo3. Both proliferating cell nuclear antigen and replication factor C enhance primer extension substantially in the cases of DNA polymerase Dpo1, Dpo3, and Dpo4 (which generate much longer extension products up to about 150-, 65-, and 150-mers, respectively) but very weakly with DNA polymerase Dpo2, which generates slightly more products of similar length up to about 80-mers. The addition of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein strongly increases DNA polymerization by Dpo1 and Dpo4, which yields extended products, mainly up to about 500-mers and 300-mers, respectively. An increase of the processivity of DNA synthesis in the presence of all accessory replication factors is the most prominent with Dpo1 and also with Dpo4
-
proliferating cell nuclear antigen
-
is greatly enhanced by the presence of proliferating cell nuclear antigen and replication factor C
-
Replication factor C
both proliferating cell nuclear antigen and replication factor C enhance primer extension substantially in the cases of DNA polymerase Dpo1, Dpo3, and Dpo4 (which generate much longer extension products up to about 150-, 65-, and 150-mers, respectively) but very weakly with DNA polymerase Dpo2, which generates slightly more products of similar length up to about 80-mers. The addition of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein strongly increases DNA polymerization by Dpo1 and Dpo4, which yields extended products, mainly up to about 500-mers and 300-mers, respectively. An increase of the processivity of DNA synthesis in the presence of all accessory replication factors (proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein) is the most prominent with Dpo1 and also with Dpo4
-
Replication factor C
-
is greatly enhanced by the presence of proliferating cell nuclear antigen and replication factor C
-
Replication factor C
-
enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner
-
single-stranded binding protein
an increase of the processivity of DNA synthesis in the presence of all accessory replication factors (proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein) is the most prominent with Dpo1 and also with Dpo4 as compared with DNA polymerase Dpo2 and Dpo3
-
single-stranded binding protein
an increase of the processivity of DNA synthesis in the presence of all accessory replication factors (proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein) is the most prominent with Dpo1 and also with Dpo4 as compared woth DNA polymerase Dpo2 and Dpo3
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.16
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.006
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.0144
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.067
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.98
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.013
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
1.22
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.0011
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.0024
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.0028
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.297
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
0.344
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.0008
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.0028
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.0032
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.007
dATP
pH 7.8, 37°C, dATP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.0088
dATP
pH 7.8, 37°C, dATP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
0.02
dATP
pH 7.8, 37°C, incorporation of dATP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
0.022
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.03
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
0.18
dATP
pH 7.8, 37°C, incorporation of dATP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.205
dATP
pH 7.5, 60°C, incorporation opposite an abasic site
0.325
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.39
dATP
pH 7.5, 37°C, DNA polymerase Dpo3
0.59
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.69
dATP
pH 7.5, 37°C, DNA polymerase Dpo4
0.79
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.88
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
1.1
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
1.2
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
2.1
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
2.2
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.000077
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.000098
dCTP
pH 7.8, 37°C, dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.0002
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
0.00044
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
0.00071
dCTP
pH 7.8, 37°C, dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
0.0041
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.0068
dCTP
-
template base: 2-fluoro-2'-deoxyinosine, pH 7.5, 37°C
0.0077
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.0084
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0098
dCTP
pH 7.5, 37°C, DNA polymerase Dpo2
0.01
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.012
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.018
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
0.02
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
0.022
dCTP
pH 7.5, 37°C, dCTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
0.026
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.027
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.039
dCTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.045
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.052
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.075
dCTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.773
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.012
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.013
dGTP
pH 7.8, 37°C, dGTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.028
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
0.049
dGTP
pH 7.8, 37°C, dGCTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.05
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.077
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.086
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.19
dGTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.25
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.43
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.47
dGTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.6
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
2.5
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.006
dTTP
pH 7.8, 37°C, dTTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.0093
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
0.0103
dTTP
pH 7.5, 37°C, dTTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
0.013
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
0.029
dTTP
pH 7.8, 37°C, dTTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.11
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.87
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.93
dTTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.94
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
1.3
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
1.6
dTTP
pH 7.5, 37°C, DNA polymerase Dpo4
3.1
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
3.7
dTTP
pH 7.5, 37°C, DNA polymerase Dpo2
3.8
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
8.9
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.223
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
0.232
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.279
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.287
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.403
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
additional information
additional information
-
pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
-
steady-state kinetics
-
additional information
additional information
-
biphasic kinetic analysis support a universal kinetic mechanism for the bypass of DNA double lesions, binding constants of the Dpo4-DNA binary complex, overview
-
additional information
additional information
-
pre-steady state kinetics, and biphasic kinetics of nucleotide incorporation at 56°C
-
additional information
additional information
-
kinetic parameters for nucleotide incorporation by Sso pol B1 exo(-) at 55°C
-
additional information
additional information
-
kinetic parameters of several elementary steps for the forward polymerization reaction
-
additional information
additional information
-
steady-state kinetic parameters for dCTP incorporation by Dpo4 T239W
-
additional information
additional information
-
steady-state kinetic parameters for next-base extension past 3-(2'-deoxy-beta-D-erythro-pentofuranosyl) pyrimido[1,2-alpha]purin-10(3H)-one
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
-
steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
-
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
-
steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
-
steady-state kinetic parameters for the single dNTP incorporation by Dpo4 T239W
-
additional information
additional information
-
biphasic dissociation kinetics of the polymerase-DNA binary complex
-
additional information
additional information
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
-
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
-
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
pH 7.5, 24°C, steady-state kinetic parameters for mispair extension by Dpo4
-
additional information
additional information
-
pH 7.5, 50°C, Km is 0.71 mM for incorporation of dTTP in a 61mer template containing N6-furfuryl-deoxyadenosine
-
additional information
additional information
steady-state kinetic parameters for next base extension from G:C and AP site:A (or C) template:primer termini
-
additional information
additional information
steady-state kinetic parameters for single-nucleotide incorporation opposite the C8- and N2-2-amino-3-methylimidazo[4,5-f]quinoline adducts of dGuo at the G3- and G1-positions of the NarI recognition sequence by Dpo4
-
additional information
additional information
-
steady-state parameters for the enzyme catalyzed insertion opposite and extension past O2-alkyl-dT
-
additional information
additional information
-
kinetic study of dNTP primer-extension opposite a benzo[a]pyrene-N2-dG-adduct with four DNA polymerases, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (about 20fold), dGTP (about 110fold) and dTTP (about 6fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNA polymerase class as eukaryotic DNA polymerase kappa and bacterial DNA polymerase IV, which accurately bypass N2-dG adducts
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.29
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.34
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.03
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.04
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.02
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.33
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.068
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.085
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
0.2
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.265
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.405
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.000016
dATP
pH 7.5, 37°C, DNA polymerase Dpo3
0.000083
dATP
pH 7.8, 37°C, incorporation of dATP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.0002
dATP
pH 7.8, 37°C, dATP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.00024
dATP
pH 7.8, 37°C, dATP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
0.00025
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-G-GAATCCTTACGAGCATCGCCCCC-3'
0.00029
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
0.0012
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0016
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.0018
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0025
dATP
pH 7.8, 37°C, incorporation of dATP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8oxoG)CACT-5'
0.0027
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.0028
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.0036
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.021
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.045
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.063
dATP
pH 7.5, 60°C, incorporation opposite an abasic site
0.073
dATP
pH 7.5, 37°C, DNA polymerase Dpo4
0.23
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.24
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.38
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.000053
dCTP
pH 7.5, 37°C, DNA polymerase Dpo2
0.00024
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
0.00027
dCTP
pH 7.8, 37°C, dCTP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.0003
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-G-GAATCCTTACGAGCATCGCCCCC-3'
0.00053
dCTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.00053
dCTP
pH 7.8, 37°C, dCTP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
0.0022
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.005
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8oxoG)CACT-5'
0.0055
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.0057
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0062
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.013
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.02
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.038
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
0.042
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
0.044
dCTP
-
template base: 2-fluoro-2'-deoxyinosine, pH 7.5, 37°C
0.056
dCTP
pH 7.5, 37°C, dCTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
0.092
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.23
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.28
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.38
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
12
dCTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.000013
dGTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.00032
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.00038
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8oxoG)CACT-5'
0.00043
dGTP
pH 7.8, 37°C, dGTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-G-GAATCCTTACGAGCATCGCCCCC-3'
0.00055
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0006
dGTP
pH 7.8, 37°C, dGTP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.00068
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.0016
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.0033
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.0045
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.0049
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.0068
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.15
dGTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.0000072
dTTP
pH 7.5, 37°C, DNA polymerase Dpo2
0.00011
dTTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.00013
dTTP
pH 7.8, 37°C, dTTP insertion opposite N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.00019
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.00029
dTTP
pH 7.8, 37°C, dTTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-G-GAATCCTTACGAGCATCGCCCCC-3'
0.0023
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0046
dTTP
-
template base: 2-bromo-2'-deoxyinosine, pH 7.5, 37°C
0.0047
dTTP
-
template base: 2-fluoro-2'-deoxyinosine, pH 7.5, 37°C
0.021
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.025
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.025
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.063
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.096
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.28
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
0.35
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
0.36
dTTP
pH 7.5, 37°C, dTTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
0.48
dTTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.012
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.028
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
0.063
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.145
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.15
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
additional information
additional information
-
pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
DNA polymerase IV (Dpo4) shows 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. Extension beyond an 8-oxoG:C pair is similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. dCTP insertion opposite 8-oxoG was lower than for opposite guanine
-
additional information
additional information
-
DNA polymerase IV (Dpo4) shows 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. Extension beyond an 8-oxoG:C pair is similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. dCTP insertion opposite 8-oxoG was lower than for opposite guanine
-
additional information
additional information
-
steady-state kinetic parameters for dCTP incorporation by Dpo4 T239W
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
-
steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
-
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
-
steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
-
steady-state kinetic parameters for the single dNTP incorporation by Dpo4 T239W
-
additional information
additional information
the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone is 1000-10000 at 37°C. The functional exonuclease proofreading active site will increase fidelity by at least 100
-
additional information
additional information
-
the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone is 1000-10000 at 37°C. The functional exonuclease proofreading active site will increase fidelity by at least 100
-
additional information
additional information
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
-
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
-
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
pH 7.5, 24°C, steady-state kinetic parameters for mispair extension by Dpo4
-
additional information
additional information
-
pH 7.5, 50°C, kcat is 0.16/s for incorporation of dTTP in a 61mer template containing N6-furfuryl-deoxyadenosine
-
additional information
additional information
steady-state kinetic parameters for next base extension from G:C and AP site:A (or C) template:primer termini
-
additional information
additional information
steady-state kinetic parameters for single-nucleotide incorporation opposite the C8- and N2-2-amino-3-methylimidazo[4,5-f]quinoline adducts of dGuo at the G3- and G1-positions of the NarI recognition sequence by Dpo4
-
additional information
additional information
-
steady-state parameters for the enzyme catalyzed insertion opposite and extension past O2-alkyl-dT
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.31
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
20.14
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
56.7
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.03
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.6
2-thio-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.016
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
25.38
5-Methyl-dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.2
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.29
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
71.4
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
168.7
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
240.9
7-deaza-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.000000041
dATP
pH 7.5, 37°C, DNA polymerase Dpo3
0.00000086
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
0.00011
dATP
pH 7.5, 37°C, DNA polymerase Dpo4
0.00046
dATP
pH 7.8, 37°C, incorporation of dATP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.001
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0016
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.0026
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.0032
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.0034
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.01
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.01
dATP
pH 7.8, 37°C, dATP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
0.02
dATP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.03
dATP
pH 7.8, 37°C, dATP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.03
dATP
pH 7.8, 37°C, dATP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
0.125
dATP
pH 7.8, 37°C, incorporation of dATP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
0.138
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.31
dATP
pH 7.5, 60°C, incorporation opposite an abasic site
75
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
235.7
dATP
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
287.5
dATP
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
0.0000054
dCTP
pH 7.5, 37°C, DNA polymerase Dpo2
0.0000071
dCTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.0095
dCTP
-
template base: 2-bromo-2'-deoxyinosine, pH 7.5, 37°C
0.026
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with O6-methylguanine as template
0.027
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.083
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.11
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.12
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.31
dCTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.7
dCTP
pH 7.8, 37°C, dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCGT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-TCAATCCTTACGAGCATCGCCCCC-3'
1.2
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCGT-G-TCAATCCTTACGAGCATCGCCCCC-3'
1.7
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
1.9
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
2.3
dCTP
pH 7.5, 37°C, dCTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
2.4
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
2.5
dCTP
pH 7.5, 37°C, dCTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
2.8
dCTP
pH 7.8, 37°C, dCTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
3.9
dCTP
pH 7.8, 37°C, dCTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
11.36
dCTP
pH 7.8, 37°C, incorporation of dCTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
23
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
28
dCTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
31
dCTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.000000068
dGTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.0000027
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.00032
dGTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.0055
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.0065
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.008
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (G) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(G)CACT-5'
0.01
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.01
dGTP
pH 7.8, 37°C, dGTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.014
dGTP
pH 7.8, 37°C, incorporation of dGTP opposite (8oxoG) in the double stranded oligonucleotide: 5'-GGGGGAAGGATTC-3'/3'-CCCCCTTCCTAAG(8ocoG)CACT-5'
0.019
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.027
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.03
dGTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.05
dGTP
pH 7.8, 37°C, dGTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.0000000019
dTTP
pH 7.5, 37°C, DNA polymerase Dpo2
0.00000012
dTTP
pH 7.5, 37°C, DNA polymerase Dpo3
0.000031
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0003
dTTP
pH 7.5, 37°C, DNA polymerase Dpo4
0.0016
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-BzG
0.0026
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-BzG
0.0028
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template: abasic site
0.0046
dTTP
-
template base: 2-bromo-2'-deoxyinosine, pH 7.5, 37°C
0.01
dTTP
pH 7.8, 37°C, dTTP insertion opposite an unmodified deoxyguanosine in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.017
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: O6-MeG
0.019
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: N2-MeG
0.02
dTTP
pH 7.8, 37°C, dTTP insertion opposite the N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion in 5'-TCAT-(N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine)-GAATCCTTACGAGCATCGCCCCC-3'
0.022
dTTP
pH 7.5, 37°C, dNTP incorporation opposite DNA lesions, template base: G
0.024
dTTP
-
template base: 2-fluoro-2'-deoxyinosine, pH 7.5, 37°C
26.9
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-(OH)2butyl-GSH in 5'-TCTC-N6dA-(OH)2butyl-GSH-GTTTATGGACCACC-3'
30
dTTP
pH 7.5, 37°C, dTTP insertion opposite the N6dA-butanetriol in 5'-TCTC-N6dA-butanetriol-GTTTATGGACCACC-3'
35
dTTP
pH 7.5, 37°C, dTTP insertion opposite an unmodified deoxyadenosine in 5'-TCTCAGTTTATGGACCACC-3'
0.04
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of a cis-syn thymine dimer of the template
0.13
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite an abasic site
0.156
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of a cis-syn thymine dimer of the template
0.54
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 3'-thymine of an nondamaged template
0.625
N1-methyl-2'-deoxyadenosine 5'-triphosphate
pH 7.5, 60°C, incorporation opposite the 5'-thymine of an nondamaged template
additional information
additional information
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pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
pre-steady-state kinetic constants for dNTP and rNTP incorporation by wild-type and F12A mutant enzyme
-
additional information
additional information
DNA polymerase IV (Dpo4) shows 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. Extension beyond an 8-oxoG:C pair is similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. dCTP insertion opposite 8-oxoG was lower than for opposite guanine
-
additional information
additional information
-
DNA polymerase IV (Dpo4) shows 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) is similar for G and 8-oxoG templates. Extension beyond an 8-oxoG:C pair is similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. dCTP insertion opposite 8-oxoG was lower than for opposite guanine
-
additional information
additional information
-
steady-state kinetic parameters for dCTP incorporation by Dpo4 T239W
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
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steady-state kinetic parameters for one-base incorporation
-
additional information
additional information
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
-
steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4
-
additional information
additional information
steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
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steady-state kinetic parameters for single-base extension reactions by Dpo4 in the presence of either Ca2+ or Mg2+
-
additional information
additional information
-
steady-state kinetic parameters for the single dNTP incorporation by Dpo4 T239W
-
additional information
additional information
transient-state kinetic analysis of Dpo4 mutants and dATP Incorporation opposite 8-oxoG.Transient-state kinetic analysis of Dpo4 mutants and dCTP incorporation opposite 8-oxoG
-
additional information
additional information
-
transient-state kinetic analysis of Dpo4 mutants and dATP Incorporation opposite 8-oxoG.Transient-state kinetic analysis of Dpo4 mutants and dCTP incorporation opposite 8-oxoG
-
additional information
additional information
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
-
kinetic for nucleotide insertion opposite template cytosine
-
additional information
additional information
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
-
kinetic parameters for single dNTP incorporation opposite template 26-mer-N-(deoxyguanosin-8-yl)-1-aminopyrene
-
additional information
additional information
pH 7.5, 24°C, steady-state kinetic parameters for mispair extension by Dpo4
-
additional information
additional information
-
pH 7.5, 50°C, kcat/Km is 0.0225/s * mM for incorporation of dTTP in a 61mer template containing N6-furfuryl-deoxyadenosine
-
additional information
additional information
steady-state kinetic parameters for next base extension from G:C and AP site:A (or C) template:primer termini
-
additional information
additional information
steady-state kinetic parameters for single-nucleotide incorporation opposite the C8- and N2-2-amino-3-methylimidazo[4,5-f]quinoline adducts of dGuo at the G3- and G1-positions of the NarI recognition sequence by Dpo4
-
additional information
additional information
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steady-state parameters for the enzyme catalyzed insertion opposite and extension past O2-alkyl-dT
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 8.5
maximal activity is observed with 20 mM Tris between pH 7 and 8.5
7.5
7.8
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
pH 7.0: about 50% of maximal activity, pH 9.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
70
70
-
the replication fidelity of the enzyme is about three times as high at 70°C as at 55°C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2 - 80
-
the incorporation rate for correct nucleotides increases by 18900fold from 2°C to 56°C, the fidelity of Dpo4 at 80°C is similar to that determined at 56°C
25 - 60
25 - 70
25 - 60
the catalytic activities of DNA polymerase Dpo2 gradually increases from 25 to 50°C but abruptly decreases at 60°C, at which it is only20% of the activity at 50°C (and even lower than the activities at 37°C) and is abolished at 70 °C
25 - 60
the catalytic activity of DNA polymerase Dpo3 gradually increases from 25 to 50°C but abruptly decreases at 60°C, at which it is 20% of the activity at 50°C (and even lower than the activity at 37°C) and is abolished at 70°C
25 - 70
activities of DNA polymerase Dpo1 gradually increases from 25 to 60°C and is substantial even at 70°C, with40% of the activity at 60°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
additional information
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replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication, kinetics and conformational dynamics by single-molecule Förster resonance energy transfer techniques, overview. The replicative polymerase can bind to DNA in at least three conformations, corresponding to an open and closed conformation of the finger domain as well as a conformation with the DNA substrate bound to the exonuclease active site of PolB1. PolB1 can transition between these conformations without dissociating from a primer-template DNA substrate. The closed conformation is promoted by a matched incoming dNTP but not by a mismatched dNTP and that mismatches at the primer-template terminus lead to an increase in the binding of the DNA to the exonuclease site
physiological function
Dpo3 is a potential player in the proper maintenance of the archaeal genome
physiological function
-
DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. This damage can be bypassed by the Y-family DNA polymerases
physiological function
-
the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100000
110000
100000
-
1 * 100000, likely it is cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
oligomer
the enzyme can form a oligomeric complex on DNA
trimer
the enzyme is able to physically associate with itself to form a trimer. This complex is stabilized in the presence of DNA. Initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. These are specific polymerase-polymerase interactions and not just separate binding events along DNA. The presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form
additional information
monomer
-
1 * 100000, likely it is cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE
additional information
Dpo4 has a spacious, highly solvent-accessible active-site region, schematic and molecular surface representations of Dpo4 ternary structure, comparison to the high-fidelity DNA polymerase Bacillus fragment, overview
additional information
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Dpo4 has a spacious, highly solvent-accessible active-site region, schematic and molecular surface representations of Dpo4 ternary structure, comparison to the high-fidelity DNA polymerase Bacillus fragment, overview
additional information
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investigation of the structure of the recombinant enzyme in the free and DNA-bound states by limited proteolysis experiments and fluorescence quenching measurements
additional information
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solution-phase NMR study, assignments for the backbone nitrogen, carbon, and amide proton NMR signals of the catalytic core of the enzyme consisting of the finger, palm, and thumb domains
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
bypass crystal structures of Dpo4:DNA(S-cdG):dCTP (error-free) and Dpo4:DNA(S-cdG):dTTP (error-prone) are catalytically incompetent. In Dpo4:DNA(S-cdG):dTTP structure, S-cdG induces a loop structure and causes an unusual 5'-template base clustering at the active site, providing the first structural evidence for the previously suggested template loop structure that can be induced by a cyclopurine lesion
crystal data and refinement parameters for the ternary (protein/DNA/GTP) complexes of Dpo4, The complexes are crystallized by sitting-drop vapor diffusion
-
crystal forms for the N249Y mutant enzyme are obtained at 22°C by the microbatch method
crystal structure of Dpo4 in complex with North-methanocarba-dATP opposite dT
crystal structure of ternary complex Dpo4/blunt-end DNA/dATP, crystals are produced by the hanging-drop method at 20°C
crystal structures of Dpo4 complexes with oligonucleotides are solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG
crystal structures of Dpo4 in complex with DNA duplexes containing the 2,4-difluorotoluene analog. The structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite 2,4-difluorotoluene and its inability to extend beyond a G:2,4-difluorotoluene pair
crystallization of an enzyme:DNA complex, sitting drop vapor diffusion method by mixing 0.001 mL of complex with 0.001 mL of a solution containing 50 mM Tris-HCl (pH 7.4 at 25°C) buffer, 12-20% polyethylene glycol 3350 (w/v), 100 mM Ca(OAc)2, and 2.5% glycerol (v/v)
crystallization of mutant enzymes R332A and R332E in complex with DNA and dGTP (R332E(8-oxoG:A), R332E(8-oxoG:C), R332A(8-oxoG:A) and R332A(8-oxoG:C)). The R332E(8-oxoG:C) structure is crystallized by the hanging drop vapor diffusion technique, using a mixture of 14% polyethylene glycol 4000 (w/v), 0.1 M calcium acetate, and 20 mM HEPES (pH 7.3) as reservoir
crystals are generated by the vapour diffusion in hanging drops. Crystal structure of the full-length enzyme Dpo4 in complex with heterodimeric sliding clamp PCNA1–PCNA2 at 2.05 A resolution. Two hinges render the multidomain polymerase flexible conformations and orientations relative to PCNA. The enzyme binds specifically to PCNA1 on the conserved ligand binding site
crystals are grown at room temperature by hanging drop vapor diffusion
crystals are grown using the sitting drop vapor diffusion method (pH 7.4 , 25°C). The Dpo4 polymerase is cocrystallized with the aflatoxin B1-formamidopyrimidine-modified template and the structure is determined at 3.0 A resolution. The structures of the ternary complexes are determined at 2.9 and 2.7 A resolutions for the dATP and dTTP complexes, respectively
determination of crystal structures of a binary Mg2+-form Dpo4–DNA complex with 1,N2-etheno-dG in the template strand as well as of ternary Mg2+-form Dpo4–DNA–dCTP/dGTP complexes with 8-oxoG in the template strand. Crystals are grown using the sitting-drop vapor-diffusion method by mixing equal amounts of Dpo4–DNA complex solution and of a reservoir solution containing 12–20% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M magnesium acetate and 20 mMTris pH 7.5
Dpo4 in complex with DNA duplex containing N2,N2-dimethyl-substituted guanine-modified template, sitting drop vapor diffusion method, using 10-15% polyethylene glycol 3350 (w/v), 30 mM NaCl, 100 mM MgCl2, and 3% glycerol (v/v)
hanging drop method, crystals of the enzyme in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide are analyzed by Raman microscopy. 13C- and 15N-labeled d*CTP, or unlabeled dCTP, are soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ and Mn2+, nucleotide incorporation is detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, is observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that is possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction does not occur. Instead, a ternary protein/template/d*CTP complex is formed
hanging-drop vapor diffusion method at 20°C, crystal structures of the enzyme in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, solved at 1.7 A and 2.1 A resolution, respectively
hanging-drop vapour-diffusion method at 21°C using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution
hanging-drop vapour-diffusion method at 21°C using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source
sitting drop vapor diffusion method, crystallization of Dpo4/(6S,8R,11S)-trans-4-hydroxynonenal-dGuo modified 18-mer template primer DNA complexes
sitting drop vapor diffusion method, crystallization of Dpo4/DNA complexes. Crystal structures reveal wobble pairing between C and O6-BzG
-
sitting drop, vapor-diffusion method with the reservoir solution containing 10–15% polyethylene glycol 3350 (w/v), 30 mm NaCl, 100 mm MgCl2, and 3% glycerol (v/v). One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine opposite template N2,N2-dimethylguanine in a post-insertion position shows dideoxycytosine folded back into the minor groove, as a catalytically incompetent complex. A second crystal has two unique orientations for the primer terminal dideoxycytosine as follows: (I) flipped into the minor groove and (II) a long pairing with N2,N2-dimethylguanine in which one hydrogen bond exists between the O-2 atom of dideoxycytosine and the N-1 atom of N2,N2-dimethylguanine, with a second water-mediated hydrogen bond between the N-3 atom of dideoxycytosine and the O-6 atom of N2,N2-dimethylguanine. A crystal structure of Dpo4 with dTTP opposite template N2,N2-dimethylguanine reveals a wobble orientation
ternary polymerase-DNA-dNTP, dGTP and/or dATP, complexes for three template-primer DNA sequences, 18-mer-primer 13-mer sequences containing 1,N2-propanodeoxyguanosine, with bound Ca2+, X-ray diffraction structure determination and analysis at 2.4-2.7 A resolution, modelling
the enzyme is mixed with DNA (1:1.2 molar ratio) in 20 mM Tris-HCl buffer (pH 8.0, 25 °C) containing 60 mM NaCl, 4% glycerol (v/v), and 5 mM 2-mercaptoethanol and then placed on ice for 1 h prior to incubation with 5 mM MgCl2 and 1 mM dGTP. Crystals are grown using the sitting drop/vapor-diffusion method with the reservoir solution containing 20 mM Tris-HCl (pH 8.0 at 25 °C), 15% polyethylene glycol 3350 (w/v), 60 mM NaCl, 5 mM MgCl2, and 4% glycerol (v/v). Two crystal structures of Dpo4 with a template N2-NaphG (in a post-insertion register opposite a 3-terminal C in the primer) are solved. One shows N2-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 “little finger” domain. The Hoogsteen face is within hydrogen bonding distance of the N4 atoms of the cytosine opposite N2-NaphG and the cytosine at the 2 position. The second structure shows N2-NaphG in an anti conformation with the primer terminus largely disordered
the structure of the enzyme bound to G*T-mispaired primer template in the presence of an incoming nucleotide is solved. As a control the structure of the enzyme bound to a matched A-T base pair at the primer terminus is also determined. The structures offer a basis for the low efficiency of the enzyme in extending a G*T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide
three crystal structures of a the enzyme in complex with Pt-GG DNA (1,2-intrastrand covalent linkage, cis-Pt-1,2-d(GpG)) at 2.9, 1.9, and 2.0 A resolution, respectively. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3'G (first insertion) and 5'G (second insertion) of Pt-GG, and the primer extension beyond the lesion site
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F12A
mutant ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA/RNA duplex product
D424A
mutation in polymerase active site, the polymerase activity is reduced more than 30fold compared to the wild-type activity
D424A/D542A
mutation in polymerase active site, complete inactivation of polymerase activity
D542A
mutation in polymerase active site, the polymerase activity is reduced more than 50fold compared to the wild-type activity
F12A
N188W
kcat/Km for dCTP is 2.9fold compared to kcat/Km of wild-type enzyme
N249Y
exhibits increased catalytic activity when compared to the wild-type enzyme, the mutation decreases the affinity for NAD(H) cofactor
R322H
the His332 mutant exhibits faster forward rate constants relative to wild-type Dpo4. The kpol values for the His332 mutant incorporation opposite G and 8-oxoG are 3.6- and 4.6fold faster than for wild-type Dpo4. The nucleotide binding affinity trend is opposite that of wild-type Dpo4, Glu332, and Leu332, with tighter dCTP binding during bypass of G. As in the case of Ala332, the kinetic analysis indicates that His332 inserts dCTP opposite G with slightly greater efficiency than opposite 8-oxoG
R322L
kpol (forward rate of polymerization) values for the Leu332 mutant incorporation opposite G and 8-oxoG are 4.1- and 1.9fold higher than those for wild-type Dpo4. The Leu332 mutant is about 2fold more efficient at incorporation of dCTP opposite 8-oxoG compared with G
R331A/R322A
mutant has lower forward rate constants relative to wild-type enzyme for both G and 8-oxoG. The double mutant-catalyzed insertion of dCTP opposite 8-oxoG is about 4fold higher than dCTP insertion opposite G. The measured binding affinity of dCTP is tighter than that of wild-type Dpo4 for unmodified DNA, but the binding affinity of dCTP opposite 8-oxoG is similar to that observed for wild-type enzyme. The catalytic efficiency for dCTP incorporation increases about 4fold for unmodified DNA and decreases about 2fold for 8-oxoG-modified DNA. The mutant fails to incorporate dATP opposite 8-oxoG in the presteady-state experiments. It inserts dCTP opposite 8-oxoG with an about 200fold greater efficiency than it does dATP and the steady-state efficiency of dATP incorporation is decreased about 27fold relative to the wild-type enzyme
R332A
mutant enzyme displays a higher affinity (lower KD,dCTP) for dCTP when bound to the unmodified DNA compared with the KD,dCTP measured for mutant-catalyzed incorporation of dCTP opposite 8-oxoG. Wild-type enzyme shows a greater affinity for dCTP opposite to 8-oxoG-modified DNA. The catalytic efficiency of the mutant is 23fold higher at incorporation of C opposite G and 1.2fold lower than wild-type enzyme in dCTP incorporation opposite 8-oxoG
R332E
mutant enzyme retains fidelity against bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) that is similar to wild enzyme. A crystal structure of the mutant and 8-oxoG:C pair reveals water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The kpol (forward rate of polymerization) value for dCTP incorporation opposite G is 4.8fold higher than for wild-type enzyme. The kpol (forward rates of polymerization) value for dCTP incorporation opposite 8-oxoG is 3.5fold higher than wild-type enzyme insertion of dCTP opposite 8-oxoG. The catalytic efficiency of the Glu332 mutant is 2.3fold greater than wild-type Dpo4 for dCTP incorporation opposite G but 1.7fold less efficient than wild-type Dpo4 for incorporation opposite 8-oxoG
T239W
Y12A
additional information
generaion of chimeras of Sulfolobus solfataricus DNA polymerase Dpo4 and Sulfolobus acidocaldarius DNA polymerase Dbh in which their little finger domains have been interchanged. Interestingly, by replacing the little finger domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. The unique but variable little finger domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member
F12A
GTP incorporation by the wild-type enzyme is about 1000fold slower than dGTP incorporation. The rate of GTP incorporation by the mutant enzyme F12A Dbh is 2-3fold slower than incorporation of dGTP. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis
F12A
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no detectable incorporation of ribonucleotide triphosphate is observed with the wild-type enzyme, the mutant form F12A efficiently incorporates and extends ribonucleotide triphosphate, even in the absence of Mn2+ ions
T239W
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the frameshift deletion is selective for purines and involves normal conformational change followed by slow phosphodiester bond formation
T239W
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the mutant enzyme is used to analyze conformational changes associated with the addition of dCTP opposite N2-alkylG adducts
T239W
kcat/Km for dCTP is 3.6fold compared to kcat/Km of wild-type enzyme
Y12A
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mutation causes an average increase of 220fold in matched ribonucleotide incorporation efficiency and an average decrease of 9fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000fold in sugar selectivity. The mutant incorporates more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both aDNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity
Y12A
active-site mutation Y12A in Dpo4, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
85
5 min, enzyme is not denatured after heating of crude cell lysate
60
70
preincubation at 70°C for 55 min minimally reduces polymerase activity
89
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Tm-value of mutant enzyme E100A: 88.9°C. Tm-value of mutant enzyme E100A/K148A: 89.4°C
90