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 found in the environment in two major forms: reduced CrIII and CrVI, or chromate. Chromate, the most biologically active species, is readily taken up by living cells and reduced intracellularly, via reactive intermediates, to stable CrIII species. CrIII, the most abundant form of chromium in the environment, does not readily cross cell membranes and is relatively inactive in vivo. However, intracellular CrIII can react slowly with both nucleic acids and proteins and can be genotoxic. We have investigated the genotoxicity of CrIII in vitro using a DNA replication assay and in vivo by CaCl2-mediated transfection of chromium-treated DNA into Escherichia coli. When DNA replication was measured on a CrIII-treated template using purified DNA polymerases (either bacterial or mammalian), both the rate of DNA replication and the amount of incorporation per polymerase binding event (processivity) were greatly increased relative to controls. When transfected into E. coli, CrIII-treated M13mp2 bacteriophage DNA showed a dose-dependent increase in mutation frequency. These results suggest that CrIII alters the interaction between the DNA template and the polymerase such that the binding strength of the DNA polymerase is increased and the fidelity of DNA replication is decreased. These interactions may contribute to the mutagenicity of chromium ions in vivo and suggest that CrIII can contribute to chromium-mediated carcinogenesis.
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PMID:A possible role for chromium(III) in genotoxicity. 193 55

Carcinogenic chromium [Cr(VI)] compounds are reduced intracellularly to DNA- and protein-reactive chromium(III) species. However, the role of Cr(III) ions in chromium-induced genotoxicity remains unclear. We have investigated the effects of chromium(III) binding on DNA replication and polymerase processivity in vitro. Chromium ions bind slowly and in a dose-dependent manner to DNA. Micromolar concentrations of free chromium inhibit DNA replication, but if the unbound chromium is removed by gel filtration, the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template is increased greater than 6-fold relative to the control. This increase is paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. These effects are optimum when very low concentrations of chromium ions are bound to the DNA [3-4 Cr(III) ions per 1000 nucleotide phosphates]. Increased concentrations of chromium lead to the production of DNA-DNA cross-links and inhibition of polymerase activity. These results suggest that low levels of DNA-bound chromium(III) ions may contribute to chromium mutagenesis and carcinogenesis by altering the kinetics and fidelity of DNA replication.
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PMID:Chromium(III) bound to DNA templates promotes increased polymerase processivity and decreased fidelity during replication in vitro. 195 61

A number of metal compounds are important environmental carcinogens; however, the molecular mechanisms of metal-induced genotoxicity are not yet understood. Chromium, for example, is substantially mutagenic in vivo and has been shown to decrease the DNA replication fidelity in vitro. But the mechanism of chromium-induced mutagenesis is unknown and the role of replication fidelity in chromium-induced carcinogenesis is unclear. We have used in vitro DNA replication assays to investigate the effects of chromium ions on DNA polymerase activity preliminary to studying their role in chromium-induced mutagenesis. Biologically active M13mp2 DNA was replicated with purified DNA polymerases in the presence of micromolar amounts of chromium with or without the normal divalent cation, magnesium. Nucleotide incorporation kinetics were determined and sequence specific pausing was analyzed by primer-extension. Our results have demonstrated an unexpected polymerase activation by low (0.5-5.0 microns) concentrations of chromium (III), although higher concentrations of chromium are increasingly inhibitory. The increased incorporation seem at low chromium(III) concentrations is the result of increased enzyme processivity and is not polymerase specific. The possible relationship between processivity and metal-ion mutagenesis is discussed.
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PMID:Effects of chromium(III) on DNA replication in vitro. 248 33

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.
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PMID:Genetic effects of chromium compounds. 634 55

The effect of three environmentally important metals, arsenic, selenium, and chromium, on the accuracy of DNA synthesis in vitro has been analyzed. The addition of arsenic to fidelity assays did not significantly alter accuracy. Selenium did not alter fidelity under normal conditions of magnesium activation, nor did it affect the mutagenicity of manganese. Chromium in the form of Cr(III) as well as Cr(VI) diminished the fidelity by which Escherichia coli DNA polymerase I copies polynucleotide templates. Nearest-neighbor analysis of the product synthesized in the presence of chromium indicates that the misincorporated in the presence of chromium indicates that the misincorporated bases are present as single-base substitutions. Chromium was also mutagenic using the recently developed phi chi 174 assay, which measures the fidelity of DNA synthesis with a natural DNA template.
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PMID:Effects of arsenic, selenium, and chromium on the fidelity of DNA synthesis. 699 86

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis. 784 91

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

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.
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PMID:Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking. 795 85

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.
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PMID:DNA polymerase arrest by adducted trivalent chromium. 814 16

Previous studies have shown that in vitro treatment of a synthetic double-stranded DNA template with chromium(III), or chromium(VI) in the presence of ascorbate, resulted in guanine-specific DNA polymerase arrests that correlated strongly with DNA-DNA cross-linking. In vivo chromium(VI) undergoes a more complicated intracellular cascade of reductive metabolism than is achievable in an in vitro model. Moreover, in living cells, DNA is highly packaged in the form of chromatin which may alter the accessibility of DNA to chromium. A repetitive primer-extension assay was employed to determine whether chromium forms polymerase-arresting lesions in vivo. Normal human lung fibroblasts treated with chromium(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucleotides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA interstrand cross-links. Genomic DNA was isolated and alphoid sequences (1-5% of the genome) were used as a substrate for repetitive primer extension using Taq polymerase. The results showed a dose-dependent, guanine-specific, replication termination, even at low doses resulting in greater than 90% survival. The same treatment resulted in dose-dependent suppression of thymidine incorporation into DNA immediately after treatment. Thymidine incorporation increased during the first 6 h after the 2-h exposure, probably related to the repair of the single strand breaks, but then returned to high suppression levels at 24 h. The chromate treatments inhibited cell growth by specific blocking of the progression of cells through S-phase of the cell cycle. The results confirmed our studies in cell-free systems and taken together they strongly indicate that guanine-guanine DNA interstrand cross-links induced by chromate in living cells is the lesion responsible for blocking DNA replication processivity.
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PMID:Chromium(VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest, DNA-DNA cross-links and S-phase blockade of cell cycle. 870 57


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