Mutational Spectrum Induced by Chromium(III) in Shuttle Vectors Replicated in Human Cells: Relationship to Cr(III)−DNA Interactions

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[[abstract]]Trivalent chromium (Cr(III)), the ultimate species of chromium(VI) intracellular reduction, can associate with DNA forming Cr(III) monoadducts and DNA-DNA cross-links. However, the mutational specificity of Cr(LII) has not been determined partly because Cr(III) has difficulty entering cells. In this study, we have characterized the types of Cr(III)-induced DNA lesions in two buffer systems and the mutational spectrum of Cr(III)-treated shuttle vectors replicated in human 293 cells. Plasmids were treated with Cr(III) in buffers consisting of either 10 mM potassium phosphate, pH 7.5 (designated as KP), or 0.2 mM Tris-HCl and 20 mu M EDTA, pH 7.4 (designated as TE/50). The amounts of Cr(III) bound to DNA increased as Cr(III) concentration increased in both buffers; these Cr(III)-DNA associations were stable in both buffers during a 24-h dialysis. The electrophoretic mobility of supercoiled DNA was markedly retarded in samples treated with Cr(III) in TE/50 but not KP buffer, suggesting that Cr(III)-mediated DNA-DNA cross-links were generated in TE/50 but did not form in KP. Polymerase-stop assay showed that DNA polymerases were mostly blocked at the 3' adjacent bases of guanines on templates treated with Cr(III) in TE/50 but were not observed on those treated in KP. The signals of Cr(III)-mediated cross-links generated in TE/50 buffers were reduced when they were dialyzed against KP buffers. Similarly, Cr(III)-DNA monoadducts formed in KP were converted to primer-template cross-links by dialysis against TE/50. The mutation frequency of Cr(III) in the supF gene of pSP189 or pZ189 shuttle vectors replicated in human cells increased as Cr(III) concentration increased in both buffers. DNA sequencing analysis showed that single-base substitutions (61-68%), two-base substitutions (3-5%), and deletions (21-34%) were induced in similar frequencies in plasmids treated with Cr(III) in either TE/50 or KP. The Cr(III)-induced base-substitution hot spots are different from those occurring spontaneously. Cr(III) enhances G . C base substitutions, particularly G . C --> C . G transversions, at 5'(G) under bar A, 5'C (G) under bar, and 5'A (G) under bar sites. Base-substitution hot spots did not correlate with strong polymerase-stop sites, suggesting that base substitutions are derived from Cr(III) monoadducts, not from DNA-DNA cross-links.[[fileno]]2050101010024[[department]]生科

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