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)

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.
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PMID:Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage. 705 30

It has been reported that single stranded viral DNA reacts with the carcinogen, chloroacetaldehyde at specific hot spots (Premaratne et al., 1993 Int. J. Biochem. 25, 1669-1672). We tested this occurrence with several other mutagens and potential carcinogens. A series of chemicals (chloroacetaldehyde, methyl, ethyl, and propyl nitro nitrosoguanidine, hydrazine, 2,4 dinitrophenyl hydrazine, hydroxylamine and methyl methanesulfonate) were each separately reacted with viral M13mp18 DNA for 2 hr at 37 degrees C and pH 4.9. The locations of adduction were identified as points of chain termination (or polymerase fall off) when the reacted DNA was subjected to a modified sequencing procedure that had ample regular labeled and unlabeled nucleotides but lacked dideoxy chain termination mixtures. Chain termination was observed to occur at specific, non-random, sites rather than with equal probability at all bases of the DNA. Chemicals with similar structures had identical points of "fall off". The pattern of chain termination appears to be unique to each class of compounds and is independent of temperature, pH, and salt concentration. Termination is believed to occur when the DNA polymerase encounters an adduct. Mutagens of different unrelated structures when reacted with this DNA produced different sites of adduct formation, while the alkyl nitro nitrosoguanidines, compounds with homologous structure showed identical points of chain termination.
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PMID:Detection of mutagen specific adduct formation in DNA using sequencing methodology. 758 13

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

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D')C proteins. The frequency of argE(ochre)-->Arg+ mutations (which occur predominantly by AT-->TA transversions) and RifS-->RifR mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.
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PMID:The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli. 780 98

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.
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PMID:The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair. 806 14

We present evidence that DNA polymerase delta of Saccharomyces cerevisiae, an enzyme that is essential for viability and chromosomal replication, is also required for base excision repair of exogenous DNA methylation damage. The large catalytic subunit of DNA polymerase delta is encoded by the CDC2(POL3) gene. We find that the mutant allele cdc2-2 confers sensitivity to killing by methyl methanesulfonate (MMS) but allows wild-type levels of UV survival. MMS survival of haploid cdc2-2 strains is lower than wild type at the permissive growth temperature of 20 degrees C. Survival is further decreased relative to wild type by treatment with MMS at 36 degrees C, a nonpermissive temperature for growth of mutant cells. A second DNA polymerase delta allele, cdc2-1, also confers a temperature-sensitive defect in MMS survival while allowing nearly wild-type levels of UV survival. These observations provide an in vivo genetic demonstration that a specific eukaryotic DNA polymerase is required for survival of exogenous methylation damage. MMS sensitivity of a cdc2-2 mutant at 20 degrees C is complemented by expression of mammalian DNA polymerase beta, an enzyme that fills single-strand gaps in duplex DNA in vitro and whose only known catalytic activity is polymerization of deoxyribonucleotides. We conclude, therefore, that the MMS survival deficit in cdc2-2 cells is caused by failure of mutant DNA polymerase delta to fill single-strand gaps arising in base excision repair of methylation damage. We discuss our results in light of current concepts of the physiologic roles of DNA polymerases delta and epsilon in DNA replication and repair.
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PMID:DNA polymerase delta is required for base excision repair of DNA methylation damage in Saccharomyces cerevisiae. 809 Jul 67

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 degrees C), but is not affected at the permissive temperature (23 degrees C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.
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PMID:DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae. 822 27

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.
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PMID:Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. 852 50

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.
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PMID:Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae. 855 Apr 96

dnaQ-encoded epsilon subunit of DNA polymerase III, possesses 3',5' exonuclease (proofreading) activity, and is a fidelity factor of polymerase III holoenzyme. It is assumed that during SOS-induced mutagenesis, UmuD', UmuC, and RecA may suppress DnaQ proofreading activity, and allow for translesional DNA synthesis at the cost of fidelity of replication. In this report SOS-dependent, MMS-induced mutagenesis and DNA repair were tested in E. coli dnaQ49 strains. Bacteria were transformed with various pDNAs harboring compilation of the umuD(D')C genes, and the influence of plasmids on mutagenesis (argE3-->Arg+) and DNA repair was tested. DNA damage and repair were tested in plasmid DNA grown in MMS-treated bacteria and isolated either immediately after MMS treatment, or after starving the cells (MFD conditions) for 30 and 60 min, then nicking activity of Fpg protein on plasmid DNAs was analyzed. It has been found that (i) repair of MMS-induced lesions depends on umuD'C, umuD' (and to much less degree, on umuDC) genes encoded in pDNA; (ii) MMS-induced mutations, in contrast to DNA repair, are highest in the cells transformed with pDNA harboring umuDC, and lowest or zero in cells with plasmids harboring umuD'C. It is postulated that UmuD'C or UmuD' proteins play a role in the repair of damaged DNA and/or in maintenance of DNA integrity. The kinetics of these processes (perhaps due to introducing too many of the lesions) seems to be different in E. coli dnaQ+ and dnaQ cells, and probably this is a reason that (iii) MMS-induced mutations in dnaQ49 strains are not subject to MFD.
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PMID:MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: contribution of UmuD' to DNA repair. 859 33


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