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)

1,N6-Ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) are formed when electrophilic vinyl chloride (VC) metabolites, chloroethylene oxide (CEO) or chloroacetaldehyde (CAA) react with adenine and cytosine residues in DNA. They were assayed for their miscoding properties in an in vitro system using Escherichia coli DNA polymerase I and synthetic templates prepared by reaction of poly(dA) and poly(dC) with increasing concentrations of CEO or CAA. Following the introduction of etheno groups, an increasing inhibition of DNA synthesis was observed. dGMP was misincorporated on CAA- or CEO-treated poly(dA) templates and dTMP was misincorporated on CAA- or CEO-treated poly(dC) templates, suggesting that epsilon A and epsilon C may miscode. The error rates augmented with the extent of reaction of CEO or CAA with the templates. Base-pairing models are proposed for the epsilon A.G. and epsilon C.T pairs. The potentially miscoding properties of epsilon A and epsilon C may explain why metabolically-activated VC and its reactive metabolites specifically induce base-pair substitution mutations in Salmonella typhimurium. Promutagenic lesions may represent one of the initial steps in VC- or CEO-induced carcinogenesis.
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PMID:Studies on the miscoding properties of 1,N6-ethenoadenine and 3,N4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis. 701 Mar 14

Chloroacetaldehyde, a rearranged metabolic product of the human carcinogen vinyl chloride, reacts with the DNA-like polymers poly(dA-dT) and poly(dC-dG) to form etheno-adducts of the adenine and cytosine bases. These treated polymers, when used as templates for E. coli DNA polymerase I in an in vitro assay, show a decreased ability to direct DNA synthesis. At the same time, increased relative levels of non-complementary nucleotides are incorporated. With the poly(dA-dT) templates 1 dGMP residue is incorporated for every approx 60 ethenoadenine residues present whilst no increased misincorporation of dCMP was detected. With the poly(dC-dG) templates 1 misincorporation of dAMP or dTMP occurred in the presence of approx 30 and 80 ethenocytosine residues respectively. A nearest neighbour analysis shows that with the modified poly(dC-dG) templates the majority of the errors were incorporated opposite cytosine (or modified cytosine) bases.
Carcinogenesis 1981
PMID:The induction of errors during in vitro DNA synthesis following chloroacetaldehyde-treatment of poly(dA-dT) and poly(dC-dG) templates. 702 22

Caffeine inhibits the activity of DNA polymerase I (E. coli) and its proteolytic large fragment in in vitro DNA replication system. DNA polymerase from Micrococcus luteus is also equally inhibited by caffeine. The extent of inhibition was more with the activated adenovirus, T4 and calf thymus DNA than with synthetic DNA template-primers. Results obtained from time-course studies indicated that caffeine inhibition reached maximum by 30 min of incubation. Enzyme kinetic studies showed that inhibition was competitive with respect to DNA template.
Carcinogenesis 1982
PMID:Caffeine inhibits DNA polymerase I from Escherichia coli: studies in vitro. 703 55

This paper describes the enzyme-catalyzed in vitro synthesis of double-stranded DNA (ds-DNA) containing [3H]-labeled O(6)-ethylguanine (O(6)-EtGua), an alkylation product strongly implicated in mutagenesis and carcinogenesis by ethylating N-nitroso carcinogens. Single-stranded DNA (ss-DNA) containing O(6)ethyl-[8-3H]-2'-deoxyguanosine was synthesized using terminal deoxynucleotidyl transferase. The parameters determining yield of reaction, base ratios, and DNA chain length, were investigated. The O(6)-EtGua-containing ss-DNA could be replicated by DNA polymerase I, as indicated by the incorporation of [8,5'-3H]-2'-deoxyguanosine-5'-monophosphate and by the resistance of the replication product to nuclease S1 digestion. ds-DNA's with chain lengths between approximately 200 and 1000 base-pairs and O(6)-EtGua/guanine molar ratios of approximately 10(-2)--10(-3) were synthesized. Their use in the analysis of enzymatic mechanisms involved in the elimination of O(6)-alkylguanine from the DNA of mammalian cells is discussed. The procedure of synthesis described for (O(6)-EtGua)-containing ds-DNA may also be applicable for the production of ds-DNA containing structurally modified bases other than O(6)-EtGua.
Carcinogenesis 1981
PMID:Enzymatic synthesis of double-stranded DNA containing radioactively labeled O(6)-ethylguanine as the only modified base. 727 13

The capacity of eukaryotic cells to modulate the activities of DNA repair enzymes during cell proliferation was examined. Using regenerating rat liver as a model system, the specific activities of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase were determined at specific intervals after partial hepatectomy. The induction of DNA replication and the stimulation of DNA polymerase were also measured in order to relate changes in the potential for DNA repair to those observed for DNA replication. As measured in nuclear extracts, the specific activities of both the uracil DNA glycosylase and the 3-methyladenine DNA glycosylase were increased in regenerating rat liver reaching maximal levels 18--24 h after partial hepatectomy. The specific activity of each DNA repair enzyme returned to basal levels by 48 h after the hepatectomy. No increase in either enzyme activity was observed in sham operated controls. The products of the reactions were identified as 3-methyladenine or as uracil by high pressure liquid chromatography or by gel filtration on Sephadex G-10. The 2--3 fold increases in the specific activity observed for each nuclear DNA repair enzyme was comparable to the 2.7 fold increase observed for DNA polymerase activity. The stimulation of DNA repair enzymes in regenerating rat liver is a further suggestion that eukaryotic cells actively regulate excision repair pathways in the defined pattern of gene expression observed during the eukaryotic cell cycle.
Carcinogenesis 1981
PMID:Induction of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase in regenerating rat liver. 727 38

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.
Carcinogenesis 1995 Aug
PMID:O6-methylguanine-induced replication blocks. 763 3

Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase beta gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase beta gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase beta gene.
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PMID:Polymorphisms in the human DNA polymerase beta gene. 770 33

A 25mer oligonucleotide containing a single N-(deoxyguanosin-8-yl-)-1-aminopyrene (dGAP), the major DNA adduct formed by reductively activated 1-nitropyrene, was synthesized. The adduct was located at nucleotide 21 from the 3' end. DNA synthesis on this template by human DNA polymerases alpha and beta, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site. Only when a 3'-->5' exonuclease-deficient Klenow fragment was used was incorporation of a nucleotide opposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent. Only a relatively small proportion of full-length product (< 5%) was detected. In the presence of Mn2+, the efficiency of bypass with this polymerase increased. When a 20mer primer was elongated in the presence of only one nucleotide triphosphate, deoxycytidylic acid was preferentially incorporated opposite the adduct. Deoxycytidine opposite the adduct was also preferred when a set of 21mer primers (containing each of the four nucleotides opposite dGAP) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these results, extension of a 15mer primer was carried out with all four deoxynucleotide triphosphates and the products were isolated. Maxam--Gilbert sequencing of each elongation product showed that primer extension occurred in an error-free manner. We conclude that dGAP is a strong block of DNA replication. However, when translesion synthesis occurs, it is largely accurate.
Carcinogenesis 1995 Apr
PMID:DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene. 772 60

We demonstrate here that stilbene estrogen (diethylstilbestrol) is converted to nuclear protein binding metabolite(s) both in vitro and in vivo. In vitro reaction of DES with nuclei from hamster liver or kidney in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES in nuclear proteins (histones; nonhistones precipitable by 2% TCA, NH2; nonhistones soluble in 2% TCA, NH30). The binding was significantly inhibited by cytochromes P450 inhibitors. In an in vitro system [3H]DES quinone, one of the metabolites of DES, was able to bind to pure nonhistone proteins RNA polymerase and DNA polymerase. The binding of [3H]DES quinone to nonhistones RNA polymerase and DNA polymerase was inhibited by low molecular weight thiols, i.e. glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid and hydroxymercuric benzoate. DES and DES metabolites inhibited transcriptional activity. In vivo [3H]DES was able to bind to nuclear proteins of hamster liver, kidneys and testes. The level of in vivo [3H]DES binding to all three types of nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was two or more fold higher than that observed in the liver or testis (nontarget organs). Four nuclear NH30 proteins (mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo binding of [3H]DES to transcriptionally active chromatin NH30 proteins also was observed. The data reported here establish that DES was able to bind to liver or kidney nuclear proteins in vitro, which was catalyzed by nuclear enzymes when fortified with an appropriate cofactor. DES quinone may be one of the protein binding metabolites. DES and DES metabolites inhibited transcriptional activity. The level of in vivo binding of [3H] DES to nuclear proteins of kidney (target organ) was double in comparison with that observed in liver or testis (nontarget organs). In vivo modifications in the chromatin proteins may be a factor in the development of DES-induced renal carcinogenesis is not clear.
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PMID:In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters. 773 58

This historical survey has shown the emergence over a period of about 60 years of a coherent view of DNA-reactive carcinogens and their effects. The earliest workers, in the 'pre-Watson-Crick' era, probably thought that the mode of action of carcinogens, then largely comprising polycyclic aromatic hydrocarbons, would be revealed through a relationship to steroid hormones, and that they would have protein receptors. This may well apply, in a broad sense, to promoting agents in carcinogenesis. Demonstration of the mutagenicity of a chemical, mustard gas, shifted attention to alkylating agents as carcinogens, and to the concept of mutagens, carcinogens and cytotoxic agents as 'radiomimetic'. Alkylating carcinogens were shown to react with DNA in vitro and in vivo in ways consistent with their action as mutagens, particularly as inducers of base substitutions, GC-->AT transitions. Carcinogenic hydrocarbons were subsequently shown to react with DNA of their target tissue, mouse skin, to extents positively correlated with their carcinogenic potency. They were found to react through aralkylating metabolites to give products that can block DNA polymerase, but can also cause base substitutions of the transversion type, mainly GC-->TA. Current interest centres on correlating the observed base substitutions that activate oncogenes or inactivate tumour suppressor genes in human cancer with the nature of exogenous and endogenous mutagens and the chemistry of their reactions with DNA, in order to deduce whether specific carcinogens can be implicated in the etiology of cancers. Ancillary to these studies are determinations of carcinogen-DNA reaction products in DNA from human sources.
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PMID:From fluorescence spectra to mutational spectra, a historical overview of DNA-reactive compounds. 780 20


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