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)

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we surveyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta, the Oct-1 POU domain, 434 Cro and the Arc repressor have a phosphate-binding module with 3D structures similar to one another. This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix, and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.
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PMID:Repetitive use of a phosphate-binding module in DNA polymerase beta, Oct-1 POU domain and phage repressors. 1022 61

DNA lesions caused by reactive oxygen species (ROS) are considered to be one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.
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PMID:Detection of tandem CC-->TT mutations induced by oxygen radicals using mutation-specific PCR. 1035 98

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.
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PMID:Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts. 1047 41

Interaction of Cr(VI) and ascorbate in vitro generates Cr(V), Cr(IV), Cr(III), carbon-based alkyl radicals, COO(*)(-), (*)OH, and ascorbate radicals and induces DNA interstrand cross-links at guanines. To determine which specific Cr species and free radicals cause DNA damage, we investigated the effects of mannitol and catalase on the formation of reactive intermediates, Cr-DNA associations, DNA polymerase-stop sites, and 8-hydroxydeoxyguanosine (8-OHdG) adducts induced by Cr(VI)/ascorbate in a Hepes buffer. EPR spectra showed that mannitol trapped reactive Cr(V), forming a stable Cr(V)-diol complex, and inhibited the radicals induced by Cr(VI)/ascorbate, whereas catalase or heat-denatured catalase enhanced the levels of Cr(V) without altering the radical signals. Mannitol markedly inhibited the retarded gel electrophoretic mobility of supercoiled plasmids and the formation of DNA polymerase-stop sites induced by Cr(VI)/ascorbate, but catalase did not. On the other hand, mannitol reduced only 32% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that Cr monoadducts (possibly DNA-Cr-mannitol adducts) are the major lesions generated in the Cr(VI)/ascorbate/mannitol/DNA solution. Native catalase but not heat-denatured catalase protected approximately 25% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that hydrogen peroxide may be involved. Mannitol could not completely inhibit the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, indicating that this DNA damage may be generated before the action of mannitol to trap Cr(V) and reactive oxygen species. Alternatively, Cr-peroxide intermediates may also lead to 8-OHdG formation to account for the incomplete prevention by mannitol. Catalase or heat-denatured catalase partially protected the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, suggesting an effect of proteins. Together, the results from this study suggest that the primary species generated during the reduction of Cr(VI) by ascorbate are hydroxyl radicals and Cr(V) species, responsible for the formation of 8-OHdG and DNA cross-links, respectively.
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PMID:Effects of mannitol or catalase on the generation of reactive oxygen species leading to DNA damage by Chromium(VI) reduction with ascorbate. 1052 78

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.
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PMID:Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta. 1093 95

Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 x 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H(2)O(2)) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of gamma-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase beta were not enhanced in expression. We also show that protection against the induction of DNA damage by H(2)O(2) in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection.
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PMID:Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment. 1102 35

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E. coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3'- or 5'-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5'-terminus of the 3' cleaved fragment, but is unable to remove DHU remaining on the 3'-terminus of the cleaved 5' fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3'-terminus of a cleaved 5' fragment, but are unable to remove DHU remaining on the 5'-terminus of a cleaved 3' fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.
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PMID:Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII. 1113 10

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.
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PMID:XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. 1116 44

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.
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PMID:Oxidation of thymine to 5-formyluracil in DNA promotes misincorporation of dGMP and subsequent elongation of a mismatched primer terminus by DNA polymerase. 1127 25

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.
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PMID:Role of the C-terminal residue of the DNA polymerase of bacteriophage T7. 1145 60


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