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Information on EC 4.2.99.B1 - DNA 5'-deoxyribose phosphate lyase and Organism(s) Homo sapiens and UniProt Accession Q9UGP5
for references in articles please use BRENDA:EC4.2.99.B1preliminary BRENDA-supplied EC number
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4.2.99.B1 DNA 5'-deoxyribose phosphate lyaseSpecify your search results
Word Map
- 4.2.99.B1
- glycosylases
- polymerases
-
abasic
-
neils
- ape1
- 8-oxoguanine
-
gap-filling
-
nei-like
-
translesion
-
8-oxog
-
base-specific
- 5-hydroxyuracil
-
long-patch
- spiroiminodihydantoin
-
y-family
The taxonomic range for the selected organisms is: Homo sapiens
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
Synonyms
beta-pol, neil2, drp lyase, polymerase iota, hsv-1 pol, 5'-drp lyase, dna beta-polymerase, atpolib, 5'-deoxyribose phosphate lyase, atpolia, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
5'-deoxyribose phosphate lyase
5'-dRP lyase
DNA polymerase beta
DNA polymerase beta deoxyribose phosphate lyase
-
DNA polymerase beta exhibiting both apurinic/apyrimidinic lyase and dRP lyase activity
dRP lyase
pol beta
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
homologous to the dRP lyase activity of polymerase beta, pol beta. Proposed to participate in single-nucleotide base excision repair in mammalian cells, which is the major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic siles in DNA
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
DNA polymerase beta (Pol beta) catalyzes both DNA synthesis at the 3'-hydroxyl terminus and esxcision of 5'-dRP moiety prior to completion of the base excsion repair (BER) by DNA ligase
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
DNA polymerase beta as the major polymerase involved in repair of oxidative base lesions, in single nucleotide replacement mechanisms. The 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
enzyme is involved in single-nucleotide base excision DNA repair, SN-BER
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
in reactions reconstituted with uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease and ligase I, pol iota can use its dRP lyase and polymerase activities to repair G-U and A-U pairs in DNA, a specialized form of base excision repair, BER. pol iota has an intrinsic dRP lyase activity and the reactions proceeds via beta-elimination involving an active residue containing a primary amine
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
reaction is part of the DNA base excision repair BER, enzyme releases 5'-terminal deoxyribose phosphate from preincised apurinic/apyrimidinic site DNA by beta-elimination
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site
the catalytic subunit of pol gamma catalyzes the release of the the 5'-deoxyribose phosphate residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The ability to trap covalent enzyme DNA complexes with NaBH4 implicates Schiff base intermediate in an beta-elimination reaction mechanism
-
SYSTEMATIC NAME
IUBMB Comments
DNA-(apurinic or apyrimidinic site) 5'-deoxy-D-ribose 5-phosphate-lyase
-
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
34-bp oligonucleotide containing uracil at position 16
? + 2-deoxy-D-ribose 5-phosphate
labelled with 3'-end by terminal deoxynucleotidyltransferase using [alpha-32P]ddATP and annealed to its complementary oligonucleotide, and pretreated with human uracil-DNA glycosylase and AP endonuclease
-
-
?
34-bp-deoxyoligonucleotide duplex containing a uracil residue at position 16
? + 2-deoxy-D-ribose phosphate
-
preincised [32P] apurinic/apyrimidinic site containing DNA, pretreated with uracil DNA-glycosylase and AP endonuclease
the reaction products are separarted by electrophoresis
-
?
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
? + 2-deoxy-D-ribose phosphate
-
in vitro single-nucleotide base excision DNA repair assay, containing amongst others uracil DNA-glycosylase, AP endonuclease and ligase I
-
-
?
49-bp oligodeoxynucleotide containing uracil at position 21
? + 2-deoxy-D-ribose 5-phosphate
-
labelled at 3'-end with [alpha-32P]ddATP by terminal deoxynucleotidyl transferase and annealed to an unlabelled complementary strand containing a G residue opposite the uracil. Before use, the substrate is treated with uracil-DNA glycosylase and AP endonuclease to generate a single nucleotide gap directly upstream from the labelled fragment containing a deoxyribose phosphate flap
-
-
?
49-residue oligonucleotide duplex DNA
? + 2-deoxy-D-ribose 5-phosphate
substrate contains uracil residue at position 21. The DNA is pretreated with uracil DNA glycosylase and AP endonuclease
-
-
?
closed-circular double-stranded DNA substrate bearing a single 8-oxoguanine/cytosine base pair at a defined position
? + 2-deoxy-D-ribose 5-phosphate
-
-
-
-
?
closed-circular double-stranded DNA substrate bearing a single dihydrouracil/guanine base pair at a defined position
? + 2-deoxy-D-ribose 5-phosphate
-
-
-
-
?
DNA with a 5'-incised apurinic/apyrimidinic site at position 21
? + 2-deoxy-D-ribose 5-phosphate
-
the uracil-DNA glycosylase-reacted DNA substrate is treated with AP endonuclease in the presence of MgCl2 to create a substrate containing a 5'-incised apurinic/apyrimidinic site. The resulting DNA substrate with a deoxyribose phosphate moiety at the 5'-end and a phosphate at the 3'-terminus is incubated with beta-pol or its amino-terminal 8-kDa domain
beta-pol is proposed to catalyze the release of the 5'-deoxyribose phosphate moiety from the cleaved apurinic/apyrimidinic site via a beta-elimination mechanism, producing 4-hydroxy-2-pentenal-5-phosphate
-
?
labeled uracil-containing DNA
? + 2-deoxy-D-ribose 5-phosphate
[32P]-labeled, treated with with uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease
-
-
?
additional information
?
-
-
reaction is part of the DNA base excision repair BER
-
-
?
additional information
?
-
in addition to removal of 5'-deoxyribose phosphate from base excision repair intermediates, enzyme also removes 5'-adenylated deoxyribose phosphate from base excision repair intermediates after abortive ligation
-
-
?
additional information
?
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site. The reaction mechanism of the lyase reaction involves a transient covalent enzyme-DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5'-dRP moiety. The Schiff base intermediate is resolved via a beta-elimination reaction, initiated by abstraction of a C2'-H atom from the 5'-deoxyribose phosphate (5'-dRP) moiety
-
-
?
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
additional information
?
-
-
reaction is part of the DNA base excision repair BER
-
-
?
additional information
?
-
catalysis of the beta-elimination of the 5' deoxyribose-5-phosphate at an abasic site in DNA where a DNA-(apurinic or apyrimidinic site) lyase has already cleaved the C-O-P bond 3' to the apurinic or apyrimidinic site. The reaction mechanism of the lyase reaction involves a transient covalent enzyme-DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5'-dRP moiety. The Schiff base intermediate is resolved via a beta-elimination reaction, initiated by abstraction of a C2'-H atom from the 5'-deoxyribose phosphate (5'-dRP) moiety
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
MgCl2
-
in reaction buffer HEPES-KOH, pH 7.5, without EDTA, enhanced enzyme activity, when 20 microM dCTP is included. No stimulation of enzyme activity with MgCl2, dCTP, and EDTA
NaCl
-
up to 50 mM no influence on the dRP lyase acticity of beta-pol or 8-kDa domain occurs. Above 200 mM the activity is completely abolished
additional information
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
EDTA
-
5 or 10 mM, inhibits beta-pol dRP lyase activity, in contrast to the 8-kDa domain dRP lyase activity that is slightly stimulated. Higher concentrations, 20 and 40 mM restore the beta-pol activity to a level similar to that in the absence of EDTA. The inhibitory effect of EDTA on the beta-pol ectivity is reversed by 25-100 mM NaCl. The same concentration has no effect on the activity of the 8-kDa domain
pyridoxal 5'-phosphate
-
2 mM, results in 95 and 75% inhibition of the dRP lyase activity of beta-pol and the 8-kDa domain, respectively. The mutant K72A of the 8-kDa domain and the full-length protein beta-pol exhibit 90 and 80% loss of activity, respectively
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
apurinic/apyrimidinic endonuclease 1
-
i.e. APE1 or AP endonulease 1, stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. 10-15% increase of activity in reactions containing both EDTA and APE1. No stimulation in presence of MgCl2. The mutant form R177A of APE1 and wild-type show similar stimulation effects on dRP lyase activity
-
dATP
-
at least 2fold enhancement of enzyme activity, maximal at 20 microM, similar results with all four deoxynucleoside triphosphates
dCTP
-
at least 2fold enhancement of enzyme activity, maximal at 20 microM, similar results with all four deoxynucleoside triphosphates. Apparent KM for dCTP on a single-nucleotide gapped DNA as substrate: 0.2 microM. It is suggested that the mechanism of stimulation may involve either Pol beta protein conformational changes upon nucleoside triphosphate binding or altered protein-DNA interactions accomanying dCMP incorporation
dGTP
-
at least 2fold enhancement of enzyme activity, maximal at 20 microM, similar results with all four deoxynucleoside triphosphates
dUTP
-
at least 2fold enhancement of enzyme activity, maximal at 20 microM, similar results with all four deoxynucleoside triphosphates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.009 - 0.0097
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
0.0005
DNA with a 5'-incised apurinic/apyrimidinic site at position 21
-
dRP lyase activity of beta-pol. To quantify the kinetic parameters for the release of deoxyribose phosphate from a preincused apurinic/apyrimidinic site-containing DNA, deoxyribose phosphate is examined as a function of apurinic/apyrimidinic site concentration. The catalytic efficiency results in a kcat/Km: 0.00015 mM/s. The catalytic efficiency of the 8-kDa amino-terminal domain is 7fold lower than that of the intact 39-kDa beta-pol
-
additional information
intact apurinic/apyrimidinic site
-
similar to that of preincised apurinic/apyrimidinic site
-
0.009
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
full-length 290-kDa Pol theta, pH 7.5, 37°C, kcat/Km: 0.047 microM/min
-
0.0097
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
98-kDa fragment of Pol theta, pH 7.5, 37°C, kcat/Km: 0.072 microM/min
-
0.0006
preincised apurinic/apyrimidinic DNA
-
pH 7.4, 37°C, in presence of 0.2 mM EDTA
-
0.007
preincised apurinic/apyrimidinic DNA
-
recombinant protein, pH 7.4, 37°C, in presence of 0.2 mM EDTA
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.007 - 0.01167
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
0.0075
DNA with a 5'-incised apurinic/apyrimidinic site at position 21
-
dRP lyase activity of beta-pol. To quantify the kinetic parameters for the release of deoxyribose phosphate from a preincused apurinic/apyrimidinic site-containing DNA, deoxyribose phosphate is examined as a function of apurinic/apyrimidinic site concentration. The catalytic efficiency results in a kcat/Km: 0.00015 mM/s. The catalytic efficiency of the 8-kDa amino-terminal domain is 7fold lower than that of the intact 39-kDa beta-pol
-
additional information
intact apurinic/apyrimidinic site
-
200fold lower compared to preincised apurinic/apyrimidinic site as substrate
-
0.007
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
full-length 290-kDa Pol theta, pH 7.5, 37°C, kcat/Km: 0.047 microM/min
-
0.01167
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
-
98-kDa fragment of Pol theta, pH 7.5, 37°C, kcat/Km: 0.072 microM/min
-
0.0043
preincised apurinic/apyrimidinic DNA
-
pH 7.4, 37°C, in presence of 0.2 mM EDTA
-
4.5
preincised apurinic/apyrimidinic DNA
-
recombinant protein, pH 7.4, 37°C, in presence of 0.2 mM EDTA
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
the activity exhibited by the 8-kDa domain is over 2 orders of magnitude lower on an intact apurinic/apyrimidinic site, as compared with a preincised apurinic/apyrimidinic site of the DNA
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
the 8-kDa-domain of pol lambda, residues 240-325 show high amino acid similarity (30%) with that of pol beta, catalyzing the same reaction, residues 1-87, UniProt-ID: P06746
UniProt
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
physiological function
additional information
study of the lyase activity of human DNA polymerase beta using analogues of the intermediate Schiff base complex
malfunction
Detection of stable DNA-protein crosslinks (DPC) between Pol beta and dL in intact cells. Formation of BER-mediated DNA-protein crosslinks, formation of oxidative polbeta-DPC in vivo. Pol beta-DPC are subsequently targeted for ubiquitylation and rapid proteolysis, which is expected to generate 5'-peptidyl-dL-DNA adducts. Inhibition of theproteasome prevents removal of Pol beta-DPC (3B), leading to their toxic accumulation with cell killing and perhaps other consequences. Mitochondrial DNA polymerase gamma (Pol gamma), which harbors a 5'-dRp lyase similar to that of Pol beta, is also trapped by 5'-dL
malfunction
three Pol beta enzymes modified at position 72 with aminooxy or hydrazinyl analogues of lysine form transient covalent bonds with the 5'-dRP moiety of the damaged DNA, in the form of an oxime or hydrazone, respectively. Both types of enzyme DNA intermediates are ultimately resolved by the lyase activities of each of the modified enzymes. The formation and resolution of these E-S complexes proceed with diminished kinetics, and with an altered pH profile compared to wild-type. Comparison of base excision repair (BER) reaction with wild-type and modified Pol beta enzymes: while the wild-type enzyme performs BER very efficiently, the BER activity of the three modified enzymes is greatly reduced. The overall BER efficiency of our modified Pol beta enzymes reflects predominantly their ability to remove the 5'-dRP group, the modified proteins demonstrate poor lyase activity
physiological function
isoform POLB expression rescues methyl methanesulfonate sensitivity in aprataxin (hnt3)- and FEN1 (rad27)-deficient yeast. Model suggests that aprataxin hydrolyzes the 5'-AMP group and allows for renewed attempts at processing of the repair intermediate, and the 5'-deoxyribose phosphate may be channeled into the normal base excision repair pathway for isoform POLB deoxyribose phsosphate removal and gap filling. In long-path base excision repair, the blocking intermediates can be processed via flap excision by FEN1. In an alternative mechanism, POLB could play a part by direct removal of the entire 5'-AMP-deoxyribose phosphate blocking group via its deoxyribose phosphate lyase activity
physiological function
DNA polymerase beta (Pol beta) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5'-deoxyribose phosphate (dRP) lyase and gap-filling DNA polymerase activities. The lyase reaction is believed to occur via a beta-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the epsilon-amino group of Lys72
physiological function
DNA polymerase beta (Pol beta) participates in mammalian base excision repair (BER). The enzyme has a two-domain architecture, reflecting its dual functionality. The polymerase activity, which replaces damaged nucleosides removed during an initial excision process, is within the C-terminal 31 kDa domain, while the N-terminal 8 kDa domain participates in a lyase function, working to remove a 5'-deoxyribose phosphate (5'-dRP) moiety from the damaged DNA substrate
physiological function
DNA polymerase beta (PolB) has both DNA polymerase and dRP lyase activities. Enzyme PolB plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER
physiological function
oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase beta (Pol beta) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (long-patch) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Pol beta cross-linked to the DNA by an amide bond. In classical base excision repair (BER), only the missing nucleotide is replaced by DNA polymerase beta (Pol beta), and the deoxyribose-5'-phosphate (5'-dRp) residue is excised by a 5'-dRp lyase activity in a discrete domain of Pol beta. The result is a nicked DNA that is competent for ligation, usually by DNA ligase III alpha. Another subpathway called long-patch BER (LP-BER) also exists, in which DNA repair synthesis replaces 2-10 nucleotides, Fen1 (flap) endonuclease excises the displaced strand, and DNA ligase I seals the nick. LP-BER repair synthesis can be initiated by Pol beta, which may (inefficiently) insert a second nucleotide. When the 5'-dRp strand is displaced by two or more nucleotides, the 5'-dRp lyase of Pol beta no longer functions, requiring Fen1 to remove the single-stranded flap. BER processing of oxidative DNA damage and the formation of DNA-protein crosslinks (DPC), overview
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). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
polymerase theta is a ca. 300 kDa polypetide. The 98-kDa C-terminal region possesses both the DNA polymerase and the dRP lyase activity. The 5'-dRP lyase activity is independent of the polymerase activity. The polymerase inactive mutant D829N/E830Q retains full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme reveals that the 5'-dRP lyase active site resides in a 24-kDa-domain
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
-
1 * 39000, consists of a 31 kDa C-terminal and a 8 kDa amino-terminal domain
additional information
additional information
-
dRP lyase activity is intrinsic to the catalytic subunit of human pol gamma
additional information
-
polymerase theta is a ca. 300 kDa polypetide. The 98-kDa C-terminal region possesses both the DNA polymerase and the dRP lyase activity. The 5'-dRP lyase activity is independent of the polymerase activity. The polymerase inactive mutant D829N/E830Q retains full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme reveals that the 5'-dRP lyase active site resides in a 24-kDa-domain
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of isoform POLB in complex with nicked DNA that represents the 5'-adenylated deoxyribose phosphate-containing base excision repair intermediate. The 5'-AMP-deoxyribose phosphate group is positioned in the lyase active site
crystallization by sitting-drop vapor diffusion. Diffraction data are obtained for crystals of Pol beta/nicked DNA complexes containing a sugar in either a closed-ring or open form, with resolution limits 2.4-2.6 A
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K310A
eliminates more than 90% of the wild-type dRP lyase activity, indicating that Lys 310 is the main nucleophile involved in the reaction, forming the Schiff base internediate during beta-elimination
D829N/E830Q
-
the polymerase inactive mutant D829N/E830Q retains full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme reveals that the 5'-dRP lyase active site resides in a 24-kDa-domain
K35A
K35A/K68A/K72A
K35R/K68R/K72R
part of the active site, above 95% loss of enzyme activity
K68A
K72A
additional information
the lyase catalytic pocket, Lys72, is replaced with each of several nonproteinogenic lysine analogues. The modified Pol beta enzymes are produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA leads to elaboration of full-length Pol beta having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine affects mainly the stability of the Schiff base intermediate and results in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine at position 72, apparently shifts the amino group to a position less favorable for Schiff base formation. This effect is attenuated when the side chain is elongated by replacing one side-chain methylene group with a bridging S atom (thialysine). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an epsilon-amino group (homoarginine) or a sterically constrained secondary amine (piperidinylalanine) leads to almost complete suppression of dRP excision activity and the ability of Pol beta to support BER
K35A
about 50% of wild-type enzyme activity, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus
K35A/K68A/K72A
devoid of dRP lyase, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus
K35A/K68A/K72A
part of the active site, above 95% loss of enzyme activity
K68A
mutation has no significant effect on the removal of 5'-AMP-deoxyribose phosphate
K72A
-
2 mM pyridoxal 5'-phosphate, the mutant K72A of the 8-kDa domain and the full-lenghth protein beta-pol exhibit 90 and 80% loss of activity, respectively
K72A
less than 10% of wild-type enzyme activity, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant pol lambda and the K310A mutant are overexpressed in Eschercihia coli and purified to near homogeneity
recombinant C-terminal 98-kDA-fragment and the mutant enzyme D829N/E830Q are purified to near homogeneity
-
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli by immobilized-metal affinity chromatography and ultrafiltration
recombinant wild-type and mutant proteins H34G, K35A, K35R, Y39F, K68A, K68R, K72A, K72R, K35A/K68A, K35A/K72A, K35A/K68A/K72A, K35R/K68R/K72R are expressed in Escherichia coli and purified
the mutants K72A, K68A/K72A, K35A, K60A, H34G, E75A, F25W, K68A, K84A, E71Q, K35A/K68A/K72A, and K68A/E71Q are expressed in Escherichia coli, and the recombinant proteins are purified to near homogeneity
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant pol lambda and the K310A mutant are overexpressed in Eschercihia coli and purified to near homogeneity
codon-optimized gene POLB, recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli
expression of mutant enzymes by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72
recombinant wild-type and mutant proteins H34G, K35A, K35R, Y39F, K68A, K68R, K72A, K72R, K35A/K68A, K35A/K72A, K35A/K68A/K72A, K35R/K68R/K72R are expressed in Escherichia coli and purified
the 98-kDa-fragment and the mutant D829N/E830Q enzyme are overexpressed in Escherichia coli BL21-CodonPlus-RP-cells
-
the mutants K72A, K68A/K72A, K35A, K60A, H34G, E75A, F25W, K68A, K84A, E71Q, and K68A/E71Q are expressed in Escherichia coli, and the recombinant proteins are purified to near homogeneity, to 95%. Mutant proteins show similar behaviour to wild-type protein, indicating less changes in structural properties
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Prasad, R.; Batra, V.K.; Yang, X.P.; Krahn, J.M.; Pedersen, L.C.; Beard, W.A.; Wilson, S.H.
Structural insight into the DNA polymerase beta deoxyribose phosphate lyase mechanism
DNA Repair
4
1347-1357
2005
Homo sapiens (P06746)
Allinson, S.L.; Dianova, II; Dianov, G.L.
DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts
EMBO J.
20
6919-6926
2001
Homo sapiens
Prasad, R.; Beard, W.A.; Chyan, J.Y.; Maciejewski, M.W.; Mullen, G.P.; Wilson, S.H.
Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5'-deoxyribose phosphate lyase activities
J. Biol. Chem.
273
11121-11126
1998
Homo sapiens (P06746)
Prasad, R.; Beard, W.A.; Strauss, P.R.; Wilson, S.H.
Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism
J. Biol. Chem.
273
15263-15270
1998
Homo sapiens
Garcia-Diaz, M.; Bebenek, K.; Kunkel, T.A.; Blanco, L.
Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polynmerase lambda. A possible role in base excision repair
J. Biol. Chem.
276
34659-34663
2001
Homo sapiens (Q9UGP5), Homo sapiens
Wong, D.; Demple, B.
Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1
J. Biol. Chem.
279
25268-25275
2004
Homo sapiens
Prasad, R.; Longley, M.J.; Sharief, F.S.; Hou, E.W.; Copeland, W.C.; Wilson, S.H.
Human DNA polymerase theta possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro
Nucleic Acids Res.
37
1868-1877
2009
Homo sapiens
Longley, M.J.; Prasad, R.; Srivastava, D.K.; Wilson, S.H.; Copeland, W.C.
identification of 5'-deoxyribose phosphate lyase activity in human DNA polymerase gamma and its role in mitochondrial base excision repair in vitro
Proc. Natl. Acad. Sci. USA
95
12244-12248
1998
Homo sapiens
Bebenek, K.; Tissier, A.; Frank, E.G.; McDonald, J.P.; Prasad, R.; Wilson, S.H.; Woodgate, R.; Kunkel, T.A.
5'-Deoxyribose phosphate lyase activity of human DNA polymerase iota in vitro
Science
291
2156-2159
2001
Homo sapiens
Caglayan, M.; Batra, V.K.; Sassa, A.; Prasad, R.; Wilson, S.H.
Role of polymerase beta in complementing aprataxin deficiency during abasic-site base excision repair
Nat. Struct. Mol. Biol.
21
497-499
2014
Homo sapiens (P06746)
Daskalova, S.M.; Bhattacharya, C.; Dedkova, L.M.; Hecht, S.M.
Probing the flexibility of the catalytic nucleophile in the lyase catalytic pocket of human DNA polymerase beta with unnatural lysine analogues
Biochemistry
56
500-513
2017
Homo sapiens (P06746)
Daskalova, S.M.; Bai, X.; Hecht, S.M.
Study of the lyase activity of human DNA polymerase beta using analogues of the intermediate Schiff base complex
Biochemistry
57
2711-2722
2018
Homo sapiens (P06746)
Quinones, J.L.; Demple, B.
When DNA repair goes wrong BER-generated DNA-protein crosslinks to oxidative lesions
DNA Repair
44
103-109
2016
Homo sapiens (P06746)
Yamamoto, R.; Umetsu, M.; Yamamoto, M.; Matsuyama, S.; Takenaka, S.; Ide, H.; Kubo, K.
AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase beta knockout mouse embryonic fibroblasts
J. Radiat. Res.
56
462-466
2015
Homo sapiens (P06746), Mus musculus (Q8K409)
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