Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

Administration of hepatocarcinogens aflataxin B1 (AFB1) and N-nitrosodimethylamine (NDMA) to rats caused single-strand breaks in hepatic nuclear DNA. The damage was found to be maximum at 4 hours following AFB1 administration and at 2 hours following NDMA administration. These damages were repaired after 17 and 4 hours, respectively in cases of AFB1 and NDMA. The activity of poly(ADP-ribose)polymerase (PARP), an enzyme known to use single-strand breaks of DNA as cofactor, was observed to increase with increasing damage to DNA and decrease as and when this damage got repaired. DNA polymerase beta and DNA ligase activities were also seen to increase and decline in a way analogous to PARP. In contrast, DNA topoisomerase activity declined corresponding to an increase in PARP activity. These observations suggest a possible role of PARP in coordinating the activities of other enzymes involved in DNA repair. It is also envisaged that these parameters can be utilized to devise strategies to counteract the deleterious effects of chemical carcinogens.
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PMID:Activity of some nuclear enzymes associated with DNA repair following hepatocarcinogen administration to rats. 759 30

Mammalian DNA polymerase beta (beta-pol), a DNA repair polymerase, is known to be constitutively expressed in cultured cells, but treatment of cells with the DNA-alkylating agents MNNG or methyl methanesulfonate has been shown to up-regulate beta-pol mRNA level. To further characterize this response, we prepared a panel of monoclonal antibodies and used one of them to quantify beta-pol in whole cell extracts by immunoblotting. We found that treatment of Chinese hamster ovary cells with either DNA-alkylating agent up-regulated the beta-pol protein level 5-10-fold. This induction appeared to be secondary to DNA alkylation, as induction was not observed with a genetically altered cell line overexpressing the DNA repair enzyme O6-methylguanine-methyltransferase. We also found that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of wild type Chinese hamster ovary cells increased expression of beta-pol protein (approximately 10-fold). Any interrelationship between this TPA response and the DNA-alkylation response was studied by treatment with combinations of MNNG and TPA. The beta-pol up-regulation observed with MNNG treatment was abrogated by TPA, and conversely the up-regulation observed with TPA treatment was abrogated by MNNG.
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PMID:Phorbol ester abrogates up-regulation of DNA polymerase beta by DNA-alkylating agents in Chinese hamster ovary cells. 760 11

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.
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PMID:Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan. 801 71

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases alpha and beta, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that had been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-fold and a 2.3-fold, respectively, in DNA polymerase beta and total DNA ligase activities in cells of the treated tumor. At 5 microM cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene and altered activities of DNA polymerases alpha and beta, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy. 812 81

A model for eukaryotic DNA damage repair is proposed in which poly(ADP-ribose) polymerase(NAD+ ADP-ribosyl transferase, EC 2,4,2,30) plays an important role. In this model, poly(ADP-ribose) polymerase regulates transcription of genes that are induced by DNA-damaging agents. This transcriptional regulation results from poly(ADP-ribosyl)ation and inactivation of DNA sequence-specific regulatory proteins such as silencer element-binding proteins, thereby inducing transcription of DNA polymerase beta, which is a DNA repair enzyme in higher eukaryotes. Poly(ADP-ribose) polymerase has a number of similarities to RecA in Escherichia coli. Therefore, the genes related to DNA damage repair in higher eukaryotes are proposed to form a "poly(ADP-ribose) polymerase regulatory network" similar to the "SOS regulatory network" in E. coli.
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PMID:Speculations on the roles of ADP-ribosyl transferase based on analogies between RecA and poly(ADP-ribose) polymerase. 824 22

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.
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PMID:Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta. 830 96


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