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)

Chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis. Chromium(VI) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable Cr(III) species. Chromium(III) forms tight complexes with biological ligands, such as DNA and proteins, which are slow to exchange. In vitro, CrCl3.6H2O primarily interacts with DNA to form outer shell charge complexes with the DNA phosphates. However, at micromolar concentrations, the Cr(III) binds to a low number of saturable tight binding sites on single-stranded M13 DNA. Additional chromium interacts in a nonspecific manner with the DNA and can form intermolecular DNA cross-links. Although high concentrations of Cr(III) inhibit DNA replication, micromolar concentrations of Cr(III) can substitute for Mg2+, weakly activate the Klenow fragment of E. coli DNA polymerase I (Pol I-KF), and act as an enhancer of nucleotide incorporation. Alterations in enzyme kinetics induced by Cr(III) increase DNA polymerase processivity and the rate of polymerase bypass of DNA lesions. This results in an increased rate of spontaneous mutagenesis during DNA replication both in vitro and in vivo. Our results indicate that chromium(III) may contribute to chromate-induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis.
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PMID:Effects of chromium on DNA replication in vitro. 784 35

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.
Carcinogenesis 1994 Oct
PMID:Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid. 795 74

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.
Carcinogenesis 1994 Nov
PMID:Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking. 795 85

A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry. Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G. st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis. The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.
Carcinogenesis 1994 Jul
PMID:Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate. 803 13

DNA polymerase beta is a nuclear protein essential to DNA repair in mammalian cells. A high frequency of mutations in this gene has been reported in colorectal cancers. To clarify the tumorigenesis steps of human prostate cancers in the molecular basis, we examined the entire coding region of the human DNA polymerase beta gene in human prostate cancer tissues using polymerase chain reaction, single-strand conformational polymorphism analysis of RNA, and sequencing analysis. Consequently, we detected DNA polymerase beta gene mutations in 2 of 12 cases (17%). The first case is an A to G transition at nucleotide 893, resulting in a substitution of the amino acid from tyrosine to cysteine. In the second case, we found an A to G transition at nucleotide 305, a T deletion at nucleotide 569, and an A insertion into the 6 repeats of A from nucleotide 612 to 617. This T deletion shifted the subsequent reading frame and resulted in the premature termination at codon 163 instead of 336. The two cases were advanced grade and stage. Present results suggest that polymerase beta gene mutations, although they occurred at relatively low frequency, are involved in certain cases of human prostate carcinogenesis.
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PMID:DNA polymerase beta gene mutation in human prostate cancer. 818 60

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.
Carcinogenesis 1994 May
PMID:The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli. 820 62

Aldehydes with specific protein-DNA crosslinking ability disrupted simian virus 40 (SV40) DNA replication to cause replication fork failure by the 40S intermediate pathway, in which replicating viral genomes become inactivated and torsionally stressed. In contrast, aldehydes without detectable protein-DNA crosslinking ability had no effect on SV40 DNA replication during the 10 min exposure times employed. This indicates that protein-DNA crosslinks block either DNA polymerase or the entire replication complex. Replication failure by the 40S pathway is known to initiate recombinational events in the damaged SV40 replicons. Similar events in cellular replicons may play a role in the clastogenic effects of formaldehyde. In addition, formaldehyde and acrolein caused accumulation of catenated (topologically linked) SV40 daughter chromosomes--a signature of topoisomerase II inhibition.
Carcinogenesis 1994 May
PMID:Aldehyde-induced protein-DNA crosslinks disrupt specific stages of SV40 DNA replication. 820 64

Twenty-eight base complementary oligonucleotides were synthesized with deoxyadenosine residues modified at the N6 position with 1-methylpyrene (MP) specifically positioned 3 bp apart in opposite DNA strands. Doubly modified constructs as well as non-modified and singly modified constructs were ligated into M13mp19 and an SV40-based shuttle vector pSVL-lac for transfection into Escherichia coli and large T-antigen-expressing monkey kidney epithelial cells respectively. Repair of MP adducts was analyzed by direct nucleotide sequencing after selection of clones containing the 28mer construct. In E. coli, double MP adducts induced base substitutions at positions mainly adjacent to modified adenines, while single MP adducts were not mutagenic. Single base insertions were also induced proximal to modified adenines. The frequency of mutation induced by double MP adducts in E. coli was approximately 4% (eight mutations out of 196 analyzed). In monkey kidney cells, double MP adducts induced one and three base deletions and single base insertions. Base substitution was observed in constructs containing non-modified and singly modified adenine residues, indicative of a significant spontaneous mutation rate. The frequency of mutation induced by double MP adducts in monkey kidney cells was approximately 9% (six mutations out of 66 clones analyzed). Modification of adenine residues by MP caused termination of DNA replication by E. coli DNA polymerase I (Klenow fragment) in vitro at the position opposite the MP adduct and at the preceding base. The repair of closely spaced polycyclic aromatic hydrocarbon adducts in opposite DNA strands is discussed as it relates to mutagenesis and carcinogenesis in mammalian cells.
Carcinogenesis 1993 Apr
PMID:Mutation in Escherichia coli and mammalian cells induced by closely spaced 1-methylpyrene-deoxyadenosine adducts in opposite DNA strands. 847 28

Formation of single strand breaks in nuclear DNA induced by hepatocarcinogens aflatoxin B1 and N-nitrosodimethylamine was observed to be more pronounced in rats maintained on a riboflavin-deficient diet compared to that on a normal diet. This increased damage was reversed on riboflavin supplementation. The induction of repair enzymes poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was significantly higher in riboflavin-deficient rats following DNA damage caused by the administration of carcinogens. Riboflavin supplementation brought down the induction to the levels found in rats maintained on normal diet. Since damage to DNA and its altered repair may relate to carcinogenesis, modulation of these parameters by riboflavin suggests a potential chemopreventive role of this vitamin.
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PMID:Modulation of carcinogen-induced DNA damage and repair enzyme activity by dietary riboflavin. 855 99

A single dose of the carcinogen aflatoxin B1 (7 mg/kg body weight) to male Wistar rats significantly enhanced the hepatic activity of protein kinase C in the particulate and nuclear fractions. The particulate fraction showed stimulation at 4 and 7 h, while the nuclear activity was increased at 17 h following administration of aflatoxin B1. The enzyme activity in cytosol revealed a significant decline corresponding to stimulation in particulate fraction. The carcinogen-activated protein kinase C stimulated autophosphorylation, and was found to accelerate in vitro phosphorylation of two model DNA synthesizing enzymes--the Klenow fragment of replicative DNA polymerase of E. Coli and a DNA primase-polymerase complex of yeast mitochondrial origin. Prior phosphorylation of these enzymes led to significant enhancement of their activities. The results imply that activation of protein kinase C and consequently the activation of DNA synthesizing enzymes may play an important role in the initiation of carcinogenesis.
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PMID:Signal transduction mechanism in response to aflatoxin B1 exposure: protein kinase C activity. 864 90


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