DNA-POLYMERASE ARREST BY ADDUCTED TRIVALENT CHROMIUM

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport sy stem and undergoes intracellular reduction to trivalent chromium, whic h strongly adducts to DNA. In this study, the effect of adducted triva lent chromium on in vitro DNA synthesis was analyzed with a polymerase -arrest assay in which prematurely terminated replication products wer e separated on a DNA sequencing gel. A synthetic DNA replication templ ate was treated with increasing concentrations of chromium(III) chlori de. The two lowest chromium doses used resulted in biologically releva nt adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparab le with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication o f the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resul ted 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 sit es occurring 1 base upstream of guanine residues on the template stran d. 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 complemen tary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template contain ing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest neverth eless did occur. Low-level, dose-dependent interstrand cross-linking b etween primer and template DNA, however, was detectable even at the lo west chromium dose analyzed. Since only 9% of chromium adducts resulte d in polymerase arrest in this system, we hypothesized that arrest occ urred when the enzyme encountered chromium-mediated interstrand DNA-DN A cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. The se results suggest that the obstruction of DNA replication by chromium -mediated DNA-DNA cross-links is a potential mechanism of chromium-ind uced genotoxicity in vivo. (C) 1994 Wiley-Liss, Inc.