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)

Human fibroblast cells treated with benzo[alpha]pyrene (BP), aflatoxin B1 (AFB1) or N-acetoxy-2-fluorenylacetamide (A-AAF) inhibited Snyder-Theilen feline sarcoma virus (ST-FeSV) focus formation. Inhibition of focus formation resulting from chemical treatment was not related to cytotoxic concentrations of chemicals in that little or not effect on cells surviving treatment was observed. Maximum inhibition of focus formation occurred with BP when the cells were treated before infection. By contrast, maximum inhibition of focus formation occurred with A-AAF and AFB1 when the cells were treated after virus infection. Inhibition of focus formation by BP and AFB1 was eliminated when virus infected cells were treated 48-96 h post-infection. While no infectious virus was detected in either chemical treated or untreated ST-FeSV virus infected cultures, comparable levels of virus-directed RNA dependent DNA polymerase enzyme assay (RDDP) activity were found in both treated and untreated cultures. The data show that the inhibitory effect on focus formation is chemically mediated while the inhibition of virus synthesis is not.
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PMID:Feline sarcoma virus in vitro infection of human cells. Influence of chemical carcinogens on focus formation. 8 Nov 13

Two diol epoxides of benzo(a)pyrene (BP), and benzo(a)pyrene 4,5-oxide, have been used to make adducts in the homopolymers polyribocytidylic acid, (rC); polyriboadenylic acid (rA), polydeoxycytidylic acid (dC) and polydeoxyadenylic acid (dA). With appropriate oligomers as primers these modified and unmodified polynucleotides were used as templates for DNA synthesis with avian myeloblastosis virus DNA polymerase (AMV) or E. coli Pol I DNA polymerase. We have found that: (1) the size of the DNA product is not markedly decreased by the presence of these these polycyclic aromatic hydrocarbon adducts in the templates; (2) the presence of adducts does not lead to increased incorporation of erroneous bases. These results, supported by kinetic data, suggest that these polymerases can bypass a site containing an adduct on the template without leaving a gap or causing misincorporation of a base and they imply that mutagenesis by BP may not be attributable to either of these mechanisms.
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PMID:Effects of benzo(a)pyrene adducts of DNA synthesis in vitro. 8 90

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells exhibited DNA excision repair when exposed to the following carcinogenic K-region epoxides: 7-methyl- and 7,12-dimethyl-benz[a]anthracene-5,6-oxide, chrysene-5,6-oxide and benzo[a]pyrene-4,5-oxide. This DNA excision repair was missing in uvr A and uvr B mutant cells. The K-region epoxide phenanthrene-9,10-oxide was ineffective in all E. coli strains tested. In contrast to the K-region epoxides which where found active only in wild type cells, 1,2,3,4-diepoxybutane and the 6,7-epoxides of the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate) elicited DNA repair in uvrA, uvrB mutant cells as well. Enzymic activities catalyzing particular repair steps were identified by determining a) repair polymerization and b) size reduction of denatured DNA. A) An easily quantifiable effect in E. coli wild type cells was epoxide-induced repair polymerization. None of the K-region epoxides tested stimulated DNA repair synthesis in uvrA, uvrB mutant cells, indicating that the uvrA-, uvrB-controlled UV-endonuclease initiated excision repair by cleaving epoxide-damaged DNA. 1,2,3,4-Diepoxybutane and the TPA-6,7-oxides induced DNA repair polymerization in uvr-deficient cells, although to a lesser extent than in wild type cells, suggesting the involvement of uvr-independent incision steps. None of the epoxides induced repair polymerization in a mutant (polA107) lacking the 5'--3'exonucleolytic activity of DNA polymerase I (exonuclease VI). The absence of any repair polymerization in the polA107 mutant indicates that the exonuclease VI plays a central role in removing epoxide-damaged nucleotides. As evidenced by greatly reduced levels of repair polymerization measured in polA1 cells, DNA polymerase I was the main polymerizing enzyme. b) As a consequence of treatment with 7-methyl-benz[a]anthracene-5,6-oxide, DNA from wild type cells, contrary to uvrA mutant cells, showed size reduction after denaturation and sedimentation in alkaline sucrose gradients. This is explained by repair-specific endonucleolytic cleavage of damaged DNA. The incision required the presence of ATP indicating that functional UV-endonuclease needs ATP as a cofactor.
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PMID:Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by carcinogenic K-region epoxides and 1,2,3,4-diepoxybutane. 15 97

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.
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PMID:DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide. 132 70

A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.
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PMID:DNA polymerase action on benzo[a]pyrene-DNA adducts. 147 43

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites.
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PMID:Lesion selectivity in blockage of lambda exonuclease by DNA damage. 169 29

We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. We show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. Polyacrylamide gels indicate that a shorter DNA product is produced on modified templates and this is confirmed by determining the average chain lengths from the ratio of chain initiations to chain elongation. Gene 4 protein primed synthesis reactions display a greater sensitivity to the presence of B[a]P adducts than do oligonucleotide-primed reactions. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, we present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies [Brown, W. C., & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754] have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, we observe an increase in synthesis but to lesser extent than observed on oligonucleotide-primed synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system. 184 52

In lung and liver cancers, p53 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrene and aflatoxin B1 which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14 nucleotide residues of the p53 gene which underwent G:C to T:A mutations in lung cancers were targeted by benzo(a)pyrene. Similarly, aflatoxin B1 formed adducts at a mutational hotspot specific for liver cancer. The same nucleotide (third base of codon 249), which mutates rarely in lung cancers, was not a target for benzo(a)pyrene. These in vitro observations indicate that p53 mutational hotspots identified in different tumors are selected targets specifically for the etiologically defined environmental carcinogens.
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PMID:Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. 193 77

We demonstrate a successful induction of DNA single strand breaks in CHO-K1 cells by cocultivation with mouse embryonic fibroblasts (MEF) during exposure to benzo(a)pyrene (BP) or 3-methylcholanthrene (MC). When compared to those induced by methyl methanesulfonate (MMS), the DNA single strand breaks induced by BP and MC were markedly accumulated by post-incubation with cytosine arabinoside (araC) and were much more delayed in their rejoining. These results suggest that the active metabolites of BP or MC produced by cocultivation with MEF or microsomal fraction (S-15) result in the formation of large DNA adducts which require an active participation of DNA polymerase alpha(delta) in the polymerization step of excision repair for their removal.
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PMID:Accumulation of polycyclic aromatic hydrocarbon-induced single strand breaks is attributed to slower rejoining processes by DNA polymerase inhibitor, cytosine arabinoside in CHO-K1 cells. 200 53

Formaldehyde treatment of human fibroblasts gave rise to DNA damage detected by a nick translation assay. This damage was not repaired by typical 'long-patch'-type excision repair as evidenced by the failure of DNA repair inhibitor post-treatment to elevate the amount of DNA strand breakage. In addition, the effects of formaldehyde on DNA repair were examined in light of a recent report suggesting that formaldehyde inhibited the repair of X-ray-induced strand breaks and UV- and benzo [a]pyrene diol epoxide-induced unscheduled DNA synthesis in human bronchial cells. We report that formaldehyde (1) was ineffective at inhibiting the sealing of X-ray- or bleomycin-induced DNA strand breaks, (2) did not inhibit the removal of pyrimidine dimers from cellular DNA at short treatment times, and (3) that the previously observed inhibition of unscheduled DNA synthesis was most likely due to the inhibition of uptake of labeled precursor into formaldehyde-treated cells. Thus, our findings are not consistent with the notion that formaldehyde inhibits the repair process in human fibroblasts. Finally, formaldehyde was shown to elevate the level of misincorporation of bases into synthetic polynucleotides catalyzed by E. coli DNA polymerase I, indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA bases.
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PMID:Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair process in human diploid fibroblasts. 241 14


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