Information on EC 4.2.99.B1 - DNA 5'-deoxyribose phosphate lyase

Word Map on EC 4.2.99.B1
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.99.B1
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
DNA 5'-deoxyribose phosphate lyase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
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
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
DNA-(apurinic or apyrimidinic site) 5'-deoxy-D-ribose 5-phosphate-lyase
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
a large dsDNA virus that encodes its own DNA replication machinery
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
25-mer-deoxyoligonucleotide 3'-labeled uracil-containing DNA duplex
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
[32P]-labeled substarte, treated with uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease
the products are stabilized by NaBH4 reduction of the Schiff base, formed between the catalytic nucleophile of the dRP lyase and C1' of rhe 2-deoxyribose 5-phosphate site, verifying the dRP removal by a beta-elimination
-
?
28-mer deoxyoligonucleotide with a 5' uracil residue
?
show the reaction diagram
-
pretreated with Eschericia coli uracil N-glycosylase
-
-
?
34-bp oligonucleotide containing uracil at position 16
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
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
show the reaction diagram
-
preincised [32P] apurinic/apyrimidinic site containing DNA, pretreated with uracil DNA-glycosylase and AP endonuclease
the reaction products are separarted by electrophoresis
-
?
34-bp-oligonucleotide containing uracil at position 16
19-bp-oligonucleotide + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
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-mer-deoxyoligonucleotide containing a uracil residue at position 16
18-mer-deoxyoligonucleotide + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
pretreated with uracil-N-glycosylase and apurunic/apyrimidinic endonuclease
-
-
?
35-bp-deoxyoligonucleotide duplex containing a uracil residue at position 15
? + 2-deoxy-D-ribose phosphate
show the reaction diagram
-
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
show the reaction diagram
-
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
show the reaction diagram
-
substrate contains uracil residue at position 21. The DNA is pretreated with uracil DNA glycosylase and AP endonuclease
-
-
?
50-bp DNA containing a single abasic site preincised with AP endonuclease
29-bp DNA fragment + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
-
-
-
-
52-pb synthetic duplex DNA
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
labelled [32P]-uracil at position 22, pretreated with uracil DNA-glycosylase and Escherichia coli endonuclease IV
the percentage of total 2-deoxyribose 5-phosphate excised is calculated by dividing the amount of the dRP lyase product formed in each reaction by the sum of this product and the amount of the substrate DNA containing intact 5'-deoxyribose-5-phosphate
-
?
closed-circular double-stranded DNA substrate bearing a single 8-oxoguanine/cytosine base pair at a defined position
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
-
-
-
?
closed-circular double-stranded DNA substrate bearing a single dihydrouracil/guanine base pair at a defined position
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
-
-
-
?
DNA with a 5'-incised apurinic/apyrimidinic site at position 21
? + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
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
show the reaction diagram
[32P]-labeled, treated with with uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease
-
-
?
mimic of a preincised DNA duplex with a 5'dRP at the site of the nick
14-mer-oligonucleotide + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
nick substrate, containing a 3'-[32P]-labelled 16-mer with a terminal 5'-uracil, which upon pretreatment with uracil-DNA glycosylase is converted to an oligonuceotide with a 5' dRP residue, having a electrophoretic mobility of a 14.5-mer
removal of 5'dRP generates a 3'-[32P]-labelled 14-mer
-
?
preincised apurinic/apyrimidinic DNA
?
show the reaction diagram
-
-
-
-
?
preincised DNA duplex with a 5'dRP flap
15-mer-oligonucleotide + 2-deoxy-D-ribose 5-phosphate
show the reaction diagram
-
flap substrate, containing a 3'-[32P]-labelled 16-mer that possesses a 5' uracil flap, which upon pretreatment with uracil-DNA glycosylase is converted to an oligonuceotide with a 5' dRP flap, having a electrophoretic mobility of a 15.5-mer
the removal of the 5' dRP generates a 3'-[32P]-labelled 15-mer
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
EDTA
-
1 mM in enzyme assay
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
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
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 hide
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 hide
LITERATURE
IMAGE
0.00021 - 0.0022
25-mer-deoxyoligonucleotide 3'-labeled uracil-containing DNA duplex
-
0.00045
28-mer deoxyoligonucleotide with a 5' uracil residue
-
pretreated with Eschericia coli uracil N-glycosylase, with concentrations 0.1-2.0 microMol, kcat/Km: 0.24 microMol/min
-
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
-
0.0018
mimic of a preincised DNA duplex with a 5'dRP at the site of the nick
-
pH 7.5, 37C
-
0.0006 - 0.007
preincised apurinic/apyrimidinic DNA
-
additional information
intact apurinic/apyrimidinic site
-
similar to that of preincised apurinic/apyrimidinic site
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0018
28-mer deoxyoligonucleotide with a 5' uracil residue
Crithidia fasciculata
-
pretreated with Eschericia coli uracil N-glycosylase, with concentrations 0.1-2.0 microMol, kcat/Km: 0.24 microMol/min
-
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
Homo sapiens
-
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
-
0.0075
mimic of a preincised DNA duplex with a 5'dRP at the site of the nick
Human herpesvirus 1
-
pH 7.5, 37C
-
0.0043 - 4.5
preincised apurinic/apyrimidinic DNA
-
additional information
intact apurinic/apyrimidinic site
Homo sapiens
-
200fold lower compared to preincised apurinic/apyrimidinic site as substrate
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
-
optimum for beta-pol dRP lyase activity
7.6
-
assay at
8 - 9
-
optimum for the 8-kDa domain dRP lyase activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
strain MCAN/ES/89/IPZ229/1/89, the enzyme activity varies with the life cycle of Leishmania infantum, being maximal in the intracellular amastigote phase
Manually annotated by BRENDA team
strain MHOM/FR/80/LEM75, the enzyme activity varies with the life cycle of Leishmania infantum, being maximal in the intracellular amastigote phase
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
containing unusual DNA, i.e. kinetoplast DNA or kDNA
Manually annotated by BRENDA team
-
8-kDa domain dRP lyase in the crude cell extract
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38200
-
NEIL2, protein part of the enzyme-DNA complex, when trapped by the addition of NaBH4 to the dRP lyase activity assay. NaBH4 reduces the Schiff base formed between the catalytic nucleophile of dRP lyase and C1' of the dRP site
39000
-
consists of a 31 kDa C-terminal and a 8 kDa amino-terminal domain
43000
-
about, wild-type mitochondrial pol beta and K72A mutant
43500
-
NEIL1, protein part of the enzyme-DNA complex, when trapped by the addition of NaBH4 to the dRP lyase activity assay. NaBH4 reduces the Schiff base formed between the catalytic nucleophile of dRP lyase and C1' of the dRP site
45000
-
1 * 45000, SDS-PAGE, wild-type mitochondrial pol beta and K72A mutant
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
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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
-
the active-site pocket of dRP lyase is formed by the residues K72, Y39, and K35. K72 directly participates in Schiff base formation on the ring-opened form of the 5'-dRP group bound at the active-site pocket. Backbone motion at the active-site residues is restricted
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native and recombinant protein
-
purified from recombinant protein of an Escherichia coli expression system
recombinant C-terminal 98-kDA-fragment and the mutant enzyme D829N/E830Q are purified to near homogeneity
-
recombinant pol lambda and the K310A mutant are overexpressed in Eschercihia coli and purified to near homogeneity
recombinant protein
recombinant protein, copurification with DNA polymerase, proved by analytical gel filtration
-
recombinant protein, expressed in Escherichia coli M15 (pREP4). The protein aggregates as inclusion bodies, solubilized in 5 M guanidinium hydrochloride and 20 mM imidazole-HCl, pH 8.0 and purified
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
expressed in Escherichia coli and purified
-
expressed in Escherichia coli BL21(DE3) via electroporation, lacking the 9-amino acid mitochondrial targeting signal sequence
-
expressed in Escherichia coli strain BL21 (DE3) competent cells
-
expressed in MB36.3 cell, wild-type, SV40 T-Ag transformed cell line haboring the lambda phage transgene lambdaLIZ; MB38DELTA4, beta-pol null, sensitive to the methylating agent methylmethanesulfonate, SV40 T-Ag transformed cell line haboring the lambda phage transgene lambdaLIZ
-
expressed in Spodoptera friguperda
-
expresses in Escherichia coli M15 (pREP4). The protein aggregates as inclusion bodies, solubilized in 5 M guanidinium hydrochloride and 20 mM inidazole-HCl, pH 8.0 and purified
recombinant pol lambda and the K310A mutant are overexpressed in Eschercihia coli and purified to near homogeneity
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
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K72A
-
PCR-based site-directed mutagenesis, deficient in forming a Schiff base intermediate and accumulating an enzyme dRP intermediate without releasing free dRP
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
E71Q
-
retains wild-type enzyme activity
E75A
-
about 75% of wild-type enzyme activity
F25W
-
about 85% of wild-type enzyme activity
H34G
-
about 45% of wild-type enzyme activity
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
K35A/K68A
-
part of the active site, similar to wild-type enzyme activity
K35A/K68A/K72A
K35A/K72A
-
part of the active site, above 95% loss of enzyme activity
K35R
-
part of the active site, no effect on enzyme activity
K35R/K68R/K72R
-
part of the active site, above 95% loss of enzyme activity
K60A
-
about 40% of wild-type enzyme activity
K68A/E71Q
-
retains wild-type enzyme activity
K68A/K72A
-
about 10% of wild-type enzyme activity
K68R
-
part of the active site, significant reduction of enzyme activity
K72R
-
part of the active site, above 95% loss of enzyme activity
K84A
-
retains wild-type enzyme activity
Y39F
-
part of the active site, similar to wild-type enzyme activity
D256A
-
site-directed mutagenesis, C-terminal polymerase domain, completely eliminates dRP lyase activity, proficient in DNA synthesis by the polymerase reaction
K35A/K68A/K72A
-
site-directed mutagenesis, amino-terminal dRP lyase domain, completely eliminates dRP lyase activity, proficient in DNA synthesis by the polymerase reaction
K35A/K68A/K72A/D256A
-
site-directed mutagenesis, amino-terminal dRP lyase domain, deficient in dRP lyase and polymerase activity
R283A
-
site-directed mutagenesis, C-terminal polymerase domain, deficient in base excision repair DNA synthesis activity of the polymerase, but has full dRP lyase activity
K552A
site-directed mutagenesis matching the active site of polbeta. The mutant Trf4K552A is overexpressed in a trf4DELTA mutant. The overproduction of Trf4 wild-type increases methylmethane sulfonate resistance similar to that of of the wild-type. The Trf4K552A mutant exhibits hypersensitivity to methylmethane sulfonate, causing double-stranded DNA breaks
additional information
-
an active-site deletion mutant of beta-pol, in which residues within the C-terminal portion, residues 263-335 that are required for DNA synthesis are deleted, completely deficient in single-nucleotide base excision repair DNA synthesis, but active in 5'-deoxyribose phosphate removal