Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.
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PMID:Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus. 83 11

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.
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PMID:DNA-repair reactions by purified HeLa DNA polymerases and exonucleases. 284 25

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.
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PMID:Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro. 608 90

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.
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PMID:Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma. 625 67

The ability of HeLa DNA polymerase alpha to utilize gapped PM2 DNAs for synthesis in a model base excision DNA repair scheme was examined. Partially depurinated PM2 DNA was incised on the 5' side of apurinic sites with HeLa apurinic/apyrimidinic endonuclease II, then the baseless sugar was removed and gaps of defined mean lengths were introduced at these sites by exonucleolytic digestion with HeLa DNase V. Gaps smaller than approximately 15 nucleotides did not serve as efficient primer-templates for DNA polymerase alpha. Gaps with mean lengths of 20-63 nucleotides did support limited DNA synthesis, but such synthesis terminated after the gap was reduced to roughly 15 nucleotides. These products were not substrates for Escherichia coli DNA ligase. In contrast, HeLa DNA polymerase beta utilize as primer-templates all of the gapped DNA substrates tested though it acted more efficiently with the smaller gaps. Moreover, the beta-polymerase was capable of filling these gaps to completion. In the case of the gaps that remained after partial closure by DNA polymerase alpha, DNA polymerase beta incorporated roughly 15 nucleotides and formed a product which was a substrate for DNA ligase. These results suggest that in vivo DNA repair pathways that involve a gap-filling DNA synthesis reaction might utilize DNA polymerase alpha only for larger gaps.
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PMID:Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme. 646 63

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.
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PMID:Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V. 684 90