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 influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).
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PMID:Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA. 167 70

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

The effect of inhibition of poly(ADP-ribose) polymerase (PARP) on the growth arrest and cell killing induced by N-methyl-N-nitrosourea (MNU) was studied in L929 fibroblasts. Depletion of NAD and ATP preceded the cell killing by a 1-h exposure to 10 or 15 mM MNU. 3-Aminobenzamide (ABA), an inhibitor of PARP, spared the depletion of NAD and ATP and prevented the cell killing. With 5 mM MNU, a depletion of NAD was promptly reversed, and there was no loss of ATP and no cell death. Aphidicolin, a DNA polymerase inhibitor, prevented the restoration of NAD, with resulting depletion of ATP and death of the cells, effects that were prevented by ABA. Azide together with 2-deoxyglucose depleted ATP, followed by a loss of NAD and cell death, changes that occurred in the absence of DNA single strand breaks (DNA SSB). ABA prevented the depletion of NAD, but not that of ATP, nor the cell killing. MNU (2.5 mM) inhibited cell growth without effect on the viability of the cells. ABA potentiated the cell growth inhibition. Thus, inhibition of PARP potentiates cell growth inhibition by limiting DNA repair mechanisms. Alternatively, inhibition of the DNA repair response to more extensive DNA damage prevents cell killing. The ATP depletion caused by poly(ADP-ribosyl)ation, rather than DNA SSB and the loss of NAD, is the more critical event in the cell killing.
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PMID:Growth inhibition and cell killing by N-methyl-N-nitrosourea: metabolic alterations that accompany poly(ADP-ribosyl)ation. 778 36

The influence of poly (ADP-ribose) polymerase (PARP) and poly ADP-ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) alpha, delta, and epsilon has been examined using the replication system containing poly(dA)4500-oligo(dT)12-18 as the template primer. PARP alone inhibited the pol activities in a dose-dependent manner even in the presence of the accessory factors for DNA pol delta, proliferating cell nuclear antigen (PCNA) and activator 1 (Al; RF-C). Both DNA pol alpha and epsilon activities were decreased approximately 10-fold under the poly ADP-ribosylating condition. In contrast, DNA synthesis by DNA pol delta holoenzyme was not affected by poly ADP-ribosylation like prokaryotic DNA pol's. The analysis of poly(dT) formed by DNA pol alpha and epsilon indicated that poly ADP-ribosylation mainly reduced the frequency of replication. These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.
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PMID:Poly (ADP-ribose) polymerase inhibits DNA replication by human replicative DNA polymerase alpha, delta and epsilon in vitro. 780 50

Homogeneously purified poly(ADP-ribose) polymerase (PARP) specifically stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The effects of PARP on DNA polymerase alpha were biphasic: at very low concentrations of DNA, it rather inhibited its activity, whereas, at higher DNA concentrations, PARP greatly stimulated it. The autopoly(ADP-ribosyl)ation of PARP suppressed both its stimulatory and inhibitory effects. By immunoprecipitation with an anti-DNA polymerase alpha antibody, it was clearly shown that PARP may be physically associated with DNA polymerase alpha. Stimulation of DNA polymerase alpha may be attributed to the physical association between the two, rather than to the DNA-binding capacity of PARP, since the PARP fragment containing only the DNA binding domain showed little stimulatory activity. The existence of PARP-DNA polymerase alpha complexes were also detected in crude extracts of calf thymus.
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PMID:Interaction of poly(ADP-ribose)polymerase with DNA polymerase alpha. 789 73

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.
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PMID:The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex. 879 42

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.
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PMID:Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9. 919 46

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.
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PMID:The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis. 935 17

The acridine derivative m-AMCA (methyl-N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), a carbamate analogue of the topoisomerase II poison amsacrine, is distinguished by its high cytotoxicity against non-cycling tumour cells. We compared the response of cultured Lewis lung carcinoma cells to m-AMCA, amsacrine and the topoisomerase I poison camptothecin. The DNA polymerase inhibitor aphidicolin reversed the cytotoxicity of camptothecin fully, that of amsacrine partially, and that of m-AMCA minimally. The ability of m-AMCA to induce the enzyme poly(ADP-ribose)polymerase (PARP) was markedly lower than that of camptothecin or amsacrine. Cell cycle responses to m-AMCA and amsacrine were similar, with slowing of progress through S-phase and arrest in G2-phase. These cell cycle changes were also observed when plateau phase cultures were exposed to drug for 1 h, washed free of drug and cultured in fresh medium, with m-AMCA having a more pronounced effect than amsacrine and camptothecin having no effect. We also examined the role of p53 protein in the response using cultured human H460 cells. Both m-AMCA and amsacrine induced p53 protein expression in proliferating but not in non-proliferating H460 cells, and induced p21WAF1 regardless of proliferation status. Both induced G1-phase cell cycle arrest. It is suggested that two cytotoxicity mechanisms can be distinguished using these drugs. The first is specific for S-phase cells, is reversed by aphidicolin and induces PARP activity. The second is cell cycle non-specific, does not induce PARP and is unaffected by aphidicolin. Camptothecin activates only the first, m-AMCA primarily the second and amsacrine activates both.
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PMID:Cellular responses to methyl-N-[4-9-acridinylamino)-2-methoxyphenyl] carbamate hydrochloride, an analogue of amsacrine active against non-proliferating cells. 938 32

Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.
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PMID:Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication. 951 81


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