Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The first pre-steady-state kinetic analysis of the stereoselectivity of a DNA polymerase, Pol beta from rat brain, toward Rp and Sp isomers of dATPalphaS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPalphaS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (k(pol)) and the apparent dissociation constant (K(d,app)) were measured with dATP, Rp-dATPalphaS, and Sp-dATPalphaS in the presence of Mg(2+), Mn(2+), or Cd(2+). The results indicate that wild type (WT) polymerase (Pol) beta can incorporate both Sp- and Rp-dATPalphaS in the presence of Mg(2+), but Sp is the preferred isomer. The stereoselectivity, defined as (k(pol)/K(d))(Sp)/(k(pol)/K(d))(Rp) (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg(2+). When Mg(2+) was substituted with Mn(2+) and Cd(2+), the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol beta complexes, as well as previous steady-state kinetic studies of other DNA polymerases. In addition, the D276R mutant was designed to introduce a potential extra hydrogen bonding interaction between the arginine side chain and the pro-Sp oxygen of the alpha-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPalphaS (Sp/Rp ratio = 1.5, 3.7, and 1.5 for Mg(2+), Mn(2+), and Cd(2+), respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPalphaS relative to WT Pol beta, suggesting that this mutant Pol beta can be used to incorporate Rp-dNTPalphaS into DNA oligomers.
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PMID:DNA polymerase beta: pre-steady-state kinetic analyses of dATP alpha S stereoselectivity and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis. 1146 64

We have identified a novel polymerase beta (Pol beta)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol beta, shows a nuclear localization that contrasts with the mitochondrial localization of Pol beta from Crithidia fasciculata, a closely related parasite, the only polymerase beta described so far in Trypanosomatidae. Li Pol beta, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol beta-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol beta, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol beta mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol beta at the infective phase of the parasite. The data suggest a role of Li Pol beta in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells.
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PMID:Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation. 1155 14

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.
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PMID:Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues. 1167 35

Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase beta or replicative polymerases delta and epsilon. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation.
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PMID:Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage. 1174 53

The DNA glycosylase pathway, which requires the sequential action of two enzymes for the incision of DNA, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and/or blocking 3'-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated. Besides the logistical problems, biological evidence hints at the existence of an alternative repair pathway. Mutants of Escherichia coli and mice (ref. 4 and M. Takao et al., personal communication) that are deficient in DNA glycosylases that remove oxidized bases are not sensitive to reactive oxygen species, and the E. coli triple mutant nei, nth, fpg is more radioresistant than the wild-type strain. Here we show that Nfo-like endonucleases nick DNA on the 5' side of various oxidatively damaged bases, generating 3'-hydroxyl and 5'-phosphate termini. Nfo-like endonucleases function next to each of the modified bases that we tested, including 5,6-dihydrothymine, 5,6-dihydrouracil, 5-hydroxyuracil and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine residues. The 3'-hydroxyl terminus provides the proper end for DNA repair synthesis; the dangling damaged nucleotide on the 5' side is then a good substrate for human flap-structure endonuclease and for DNA polymerase I of E. coli.
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PMID:Alternative nucleotide incision repair pathway for oxidative DNA damage. 1180 38

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f(ins) = V(max)/K(m) for insertion) and extension (f(ext) = V(max)/K(m) for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) dTMP (2.4 x 10(-4)) approximately dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.
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PMID:Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics. 1183 60

We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Franklin, M., Wang, J., and Steitz T. (2001) Cell 105, 657-667] was consistent with the following sets of interactions between the conserved residues and oxygen atoms in the triphosphate portion of the incoming dNTP: (i) the epsilon-amino group of K560 contacts oxygen atoms of the alpha- and gamma-phosphates, (ii) the amide side chain of Asn 564 forms a hydrogen bond via a water molecule with the nonbridging oxygen of the beta-phosphate, and (iii) the epsilon-amino and delta-guanidino groups of K486 and R482, respectively, contact the nonbridging oxygens of the gamma-phosphate. We have also determined the pre-steady-state kinetic parameters for the addition of both dCTP and dCDP onto a 13/20mer primer/template with an exo(-) derivative of RB69 DNA polymerase and have shown that the deoxynucleoside diphosphate can be incorporated, in contrast to the behavior of the Klenow fragment which cannot use dCDP as a substrate. We have shown that, with RB69 DNA polymerase, in contrast to the Klenow fragment, there is no inhibition of the primer-extension reaction by incoming NTPs having either noncomplementary bases or ribo- instead of a deoxyribose moieties. This implies that the mode of recognition of incoming dNTPs and triggering of the conformational change, which is thought to occur prior to the chemical step, differs between these two enzymes.
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PMID:Correlation of the kinetics of finger domain mutants in RB69 DNA polymerase with its structure. 1185 99

In Escherichia coli the mutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species. The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitro the hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate. Thus, the protein is thought to "sanitize" the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic. However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model. We have devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts of three different mutT mutants of E. coli, even after growth of the bacteria in the presence of hydrogen peroxide. Our estimated upper limit for 8-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 microm. When 8-oxo-dGTP was added at 0.34 microm to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors. These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein.
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PMID:Assessing the metabolic function of the MutT 8-oxodeoxyguanosine triphosphatase in Escherichia coli by nucleotide pool analysis. 1185 56

Cells that depend on oxygen for survival constantly produce reactive oxygen species that attack DNA to produce a variety of lesions, including single-strand breaks with 3'-blocking groups such as 3'-phosphate and 3'-phosphoglycolate. These 3'-blocking ends prevent the activity of DNA polymerase and are generally removed by DNA repair proteins with 3'-diesterase activity. We report here the purification and partial characterization of a 45 kDa protein from Schizosaccharomyces pombe total extract based on the ability of this protein to process bleomycin- or H(2)O(2)-damaged DNA in vitro to allow DNA repair synthesis by DNA polymerase I. Further analysis revealed that the 45 kDa protein removes 3'-phosphate ends created by the Escherichia coli fpg AP lyase following the incision of AP site but is unable to process the 3'-alpha,beta unsaturated aldehyde generated by E. coli endonuclease III. The protein cannot cleave DNA bearing AP sites, suggesting that it is not an AP endonuclease or AP lyase. We conclude that the 45 kDa protein purified from S. pombe is a DNA 3'-phosphatase.
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PMID:Purification and partial characterization of a DNA 3'-phosphatase from Schizosaccharomyces pombe. 1205

The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase gamma (pol gamma) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol gamma was a possible target of ROS with H2O2 and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 microM H2O2 significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H2O2 treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol gamma resulting from H2O2 treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol gamma was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol gamma as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.
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PMID:The mitochondrial DNA polymerase as a target of oxidative damage. 1208 65


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