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)

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase alpha, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase alpha and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.
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PMID:DNA damage-induced replication arrest in Xenopus egg extracts. 1458 53

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.
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PMID:Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents. 1464 38

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.
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PMID:UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication. 1473 87

DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.
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PMID:Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping. 1525 44

In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).
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PMID:Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance. 1532 99

During a survey of plant secondary metabolites for DNA polymerase beta lyase inhibitors, we found that a crude methyl ethyl ketone extract prepared from Maytenus putterlickoides showed strong inhibition of the lyase activity of DNA polymerase beta in an in vitro assay. Bioassay-guided fractionation of the extract, using an in vitro assay, resulted in the discovery of a new active principle, 30-(4'-hydroxybenzoyloxy)-11alpha-hydroxylupane-20(29)-en-3-one (1), as well as a known compound, (-)-epicatechin (2). Compounds 1 and 2 exhibited DNA polymerase beta lyase inhibitory activity with IC50 values of 62.8 and 18.5 microM, respectively. Compound 2 was capable of potentiating the action of the monofunctional methylating agent methyl methanesulfonate in cultured human cancer cells, consistent with the possible utility of inhibitors of this type in vivo.
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PMID:DNA polymerase beta lyase inhibitors from Maytenus putterlickoides. 1549 54

DNA polymerase (Pol) beta null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol beta, SSB accumulate in G1 phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol beta null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.
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PMID:The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells. 1564 10

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.
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PMID:Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure. 1570 27

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2Delta and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1(+), which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase alpha. Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.
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PMID:Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair. 1591 39

The DNA polymerase beta (Pol beta) null background renders mouse embryonic fibroblast (MEF) cells base excision repair deficient and hyper-mutagenic upon treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS). This effect involves an increase in all types of base substitutions, with a modest predominance of G to A transitions. In the present study, we examined the hypothesis that the MMS-induced mutagenesis in the Pol beta null MEF system is due to a lesion bypass mechanism. We studied the effect of RNAi mediated down-regulation of the lesion bypass factor REV1. The steady-state level of REV1 protein was reduced by more than 95% using stable expression of a siRNA construct in a Pol beta null cell line. We found that REV1 expression is required for the MMS-induced mutagenesis phenotype of Pol beta null MEF cells. In contrast, cell survival after MMS treatment is not reduced in the absence of REV1.
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PMID:REV1 mediated mutagenesis in base excision repair deficient mouse fibroblast. 1595 May 50


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