Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA single-strand breaks are caused by aqueous extracts of cigarette tar, due to the reduction of oxygen to superoxide by tar and the subsequent production of hydroxyl radicals. The action of DNA metabolism enzymes on these single-strand breaks has been studied to probe the consequences of these lesions for DNA repair. Our results demonstrate that cigarette tar-induced nicks are blocked at the 3' terminus since they are totally incapable of activating DNA for DNA synthesis by Escherichia coli DNA polymerase I. The 3' termini of these tar-induced nicks are activated, however, for DNA synthesis by E. coli exonuclease III or by the 3' phosphatase activity of T4 polynucleotide kinase. Because of the inability of tar-induced lesions to support DNA synthesis, they probably require a multi-step process for repair in vivo. As a consequence, the overall likelihood of mutation is increased due to the possibility for error at each step of the repair process.
Carcinogenesis 1987 Oct
PMID:DNA synthesis is blocked by cigarette tar-induced DNA single-strand breaks. 282 Jun 3

Novobiocin inhibits DNA topoisomerases. It also inhibits excision repair of DNA photodamage, blocking both repair synthesis and the earlier step of incision at u.v. damage sites (as measured by the accumulation of DNA strand breaks in u.v.-irradiated interphase cells treated with DNA synthesis inhibitors such as hydroxyurea or cytosine arabinoside). It has been supposed, therefore, that novobiocin affects repair by blocking a putative topoisomerase step prior to incision. But we find that novobiocin also has a marked dose- and time-dependent effect on mitochondria: in cells exposed to novobiocin, mitochondria swell and their cristae become disrupted, and the intracellular ATP:ADP ratio is lowered, though the membrane potential is maintained as judged by rhodamine 123 fluorescence. Mitotic cells are more resistant to mitochondrial disruption by novobiocin than are interphase cells. This correlates with a relative resistance of u.v.-irradiated mitotic cells to the inhibition of incision by novobiocin. The chromosomal decondensation that results from the accumulation of DNA breaks due to incision when u.v.-irradiated mitotic cells are treated with hydroxyurea and cytosine arabinoside is largely suppressed by novobiocin. Furthermore, the suppression of induced strand break accumulation is partly due to a suppression by novobiocin of the uptake and phosphorylation of cytosine arabinoside; breaks accumulated in u.v.-irradiated cells in the presence of aphidicolin, an inhibitor of DNA polymerase alpha that does not require phosphorylation, are less novobiocin-sensitive. We conclude that the effects of novobiocin on excision repair are more likely to be due to a non-specific effect on ATP metabolism than to a specific effect on a repair-related topoisomerase.
Carcinogenesis 1985 Sep
PMID:Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases. 299 34

We have demonstrated that carcinogen damage to DNA induces the production of cellular factors that act in trans to enhance the asynchronous replication of polyoma viral DNA. Exposure of a polyoma virus-transformed rat cell line to benzo[a]pyrene-7,8-diol-9,10-oxide (BPDE), the ultimate carcinogenic metabolite of benzo[a]pyrene, led to the accumulation of heterogeneously sized free viral DNA molecules which contain polyoma origin sequences as well as cellular sequences that flank the integrated viral DNA. When the sequence gpt was linked to the polyoma early region and transfected into rat cells, it underwent asynchronous replication in response either to direct treatment of the transfected cells with BPDE, or to fusion of untreated transfected cells with normal cells previously exposed to BPDE. Transient arrest of the cell cycle by hydroxyurea, isoleucine deprivation or methotrexate caused a slight enhancement of viral DNA replication when compared with BPDE. Both aphidicolin, an inhibitor of DNA polymerase alpha, and 3-aminobenzamide, an inhibitor of poly[ADP]ribosyl transferase, caused marked inhibition of BPDE-induced viral DNA synthesis. The induction of a trans-acting factor in response to damage of cellular DNA may be relevant to synergistic interactions between environmental chemicals and DNA viruses in cell transformation and to the general phenomenon of gene amplification.
Carcinogenesis 1986 Jun
PMID:Carcinogen induced asynchronous replication of polyoma DNA is mediated by a trans-acting factor. 301 4

The rate of intracellular ligation of excision repair patches has been measured under conditions of inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide. Excision repair patches in DNA of cells damaged by methyl methanesulfonate were labeled with [3H]thymidine and blocked at an intermediate stage by aphidicolin, an inhibitor of DNA polymerase alpha. Nearly half of the [3H]thymidine label in the repair patches was sensitive to rapid digestion by exonuclease III, indicating that the label was at unligated 3' termini of repair sites. Removal of [3H]thymidine and aphidicolin permitted the intracellular ligation rate to be determined. From analysis of chromatin, ligation appeared to occur rapidly, independent of the effect of 3-aminobenzamide. Analysis of purified DNA, however, indicated that high doses of methyl methanesulfonate resulted in slow ligation rates but that 3-aminobenzamide accelerated the rates of ligation. The analysis of chromatin, therefore, indicates that unligated repair sites are sites of protein accretion which block exonuclease III action. The results from analysis of DNA indicate that poly(ADP-ribose) synthesis and associated pool depletion inhibits ligation rates; 3-aminobenzamide prevents poly(ADP-ribose) synthesis, maintains pool levels high and facilitates rapid ligation.
Carcinogenesis 1986 Jun
PMID:DNA ligation and changes in chromatin structure associated with repair patches under conditions of inhibition of poly(ADP-ribose) synthesis. 308 71

The data in this paper show that when the inhibition of growth is measured, xeroderma pigmentosum (XP) complementation groups A, G and D are very sensitive to 4-nitroquinoline-1-oxide (4NQO), whereas only XP groups G and D are very sensitive to 3-methyl-4NQO (3me4NQO). Cells belonging to XP-C group are not particularly sensitive to either agent. Thus there are different epistasis groups for the excision repair of DNA adducts induced by these agents as opposed to the repair of u.v. damage. DNA polymerase alpha is involved in the repair of 4NQO-induced lesions because aphidicolin blocks their repair. XP cells from all the above groups are defective to some extent in this repair. The degree of repair defectiveness follows that seen after u.v., with even the XP-C cell line used having reduced repair (despite the fact that the inhibition of growth by 4NQO in this cell line was not markedly different from normal). Aphidicolin did not induce breaks in the normal or XP cell lines exposed to 3me4NQO, thus the repair of lesions induced by 3me4NQO does not involve DNA polymerase alpha in any of the cell lines. Finally, catalase reduces the alkaline labile lesions induced by 4NQO, but not 3me4NQO, suggesting the latter agent does not induce substantial amounts of DNA damage by the generation of radicals.
Carcinogenesis 1987 Aug
PMID:The response to DNA damage induced by 4-nitroquinoline-1-oxide or its 3-methyl derivative in xeroderma pigmentosum fibroblasts belonging to different complementation groups: evidence for different epistasis groups involved in the repair of large adducts in human DNA. 311 41

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.
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PMID:O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication. 345 56

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.
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PMID:Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis. 346 67

To test whether vinyl chloride-induced mutagenesis might involve ambiguous base pairing of 1,N6-etheno-adenine (epsilon A) during DNA synthesis, we examined the base pairing potential of epsilon dATP during DNA synthesis catalyzed by Escherichia coli DNA polymerase I (Klenow fragment). An electrophoretic assay of chain elongation was used to assess the degree to which epsilon dATP could substitute for each of the normal dNTPs during elongation of a primer annealed to a bacteriophage template. Despite the fact that the etheno bridge completely blocks normal Watson-Crick pairing of epsilon A with T, we observed that epsilon dATP could substitute for dATP during primer elongation (although inefficiently). In addition, detectable substitution of epsilon dATP for dGTP and dCTP occurred, indicating that epsilon A exhibits ambiguous base pairing properties. The relative ease of epsilon dAMP incorporation (opposite template T, C and G) appeared to vary considerably at different positions along the template. The major form of epsilon A incorporation (replacement of A) was confirmed by measurements of epsilon dATP----epsilon dAMP turnover (a commonly used method for detecting misincorporation), and also by the demonstration that epsilon A was present in enzymatic hydrolysates prepared from DNA that was synthesized with epsilon dATP replacing dATP. A model for ambiguous base pairing of epsilon dATP is proposed, in which incorporation occurs via the protonated, syn form of epsilon dATP.
Carcinogenesis 1986 Sep
PMID:Utilization of 1,N6-etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli. 352 67

It has been shown previously that deoxyguanosine residues in DNA are hydroxylated at the C-8 position both in vitro and in vivo to produce 8-hydroxydeoxyguanosine (8-OH-dG) by various agents that produce oxygen radicals such as reducing reagents-O2, metal ions-O2, polyphenol-H2O2-Fe3+, asbestos-H2O2 or ionizing radiation. These agents are mostly either mutagenic or carcinogenic; therefore, the formation of 8-OH-dG can also be considered a likely cause of mutation or carcinogenesis by oxygen radicals. It is of interest to know whether the 8-OH-dG residue in DNA is misread during DNA replication. To answer this question, we have examined the effect of the 8-OH-dG residue in DNA on the fidelity of DNA replication using a DNA synthesis system in vitro with Escherichia coli DNA polymerase I (Klenow fragment). The synthetic oligodeoxynucleotides, with or without an 8-OH-dG residue in a specified position, were chemically synthesized and used as templates for DNA synthesis under the conditions of the dideoxy chain termination sequencing method. Surprisingly, in addition to misreading of the 8-OH-dG residue itself, pyrimidines next to the 8-OH-dG residue (G has not yet been tested) were also misread.
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PMID:Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. 357 69

In permeable human fibroblasts, novobiocin, coumermycin and nalidixic acid completely inhibit u.v.-induced DNA repair synthesis, with 50% inhibition occurring at 500, 24 and 8800 microM respectively. Novobiocin also inhibits damage-specific incision of DNA in u.v.-irradiated permeable human fibroblasts by at least 75%. It has been suggested that effects of novobiocin on DNA excision repair result from changes in ATP pools; this explanation is not applicable to our data because excision repair in the permeable cell system is entirely dependent on exogenous ATP. It has also been suggested that novobiocin-induced inhibition of repair is mediated by alterations of chromatin structure recognizable by electron microscopy as gross chromatin clumping. There were no ultrastructural alterations, however, in the nuclei of permeable cells that had been incubated with 1 mM novobiocin. We conclude that, in human cells, novobiocin, coumermycin, and nalidixic acid directly inhibit the excision repair of u.v. damage to DNA, and that one locus of inhibition lies at or before the incision step. Because 1 mM novobiocin completely abolishes u.v.-induced repair synthesis in permeable cells, but inhibits damage-specific incision by only 75%, there seems to be a second site of inhibition following the incision step. The similarity between the concentrations of novobiocin, nalidixic acid and coumermycin required to inhibit u.v.-induced excision repair and the concentrations required to inhibit human DNA polymerase alpha suggest that the distal locus of inhibition may be DNA polymerase alpha-mediated repair patch synthesis. The proximal inhibitory site may be a type II DNA topoisomerase.
Carcinogenesis 1987 Jun
PMID:Direct inhibition of u.v.-induced DNA excision repair in human cells by novobiocin, coumermycin and nalidixic acid. 360 79


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