The goals of this research are two-fold: to determine the mechanisms of mutagenesis induced by the carcinogenic metals nickel and chromium and to use Ni(II) and Cr(III) ions as tools to investigate the enzymatic mechanisms responsible for DNA polymerase function and fidelity. Although the mechanisms of carcinogenesis induced by these metals are complex and still largely unknown, both metals can produce DNA base damage (via the intracellular production of active oxygen species) and both Ni(II) and Cr(III) can interact with DNA polymerases in vitro in a biphasic manner to enhance and inhibit polymerase function and fidelity. Do these metal ions also enhance the mutagenic potential of other forms of DNA damage and can they induce mutagenesis by the direct inhibition of polymerase function? The specific aims of this research are: to establish whether either 1) Cr(III) or 2) Ni(II) can alter the kinetics and fidelity of DNA replication past a model mutagenic lesion, O6-methyl guanine; 3) to investigate the effects of these metal ions on the kinetics of DNA polymerase 3'-5' exonuclease function; 4) to determine whether Cr(III) affects the use of deoxynucleotide triphosphate substrates by forming a beta, gamma-Cr(III)-dNTP complex and/or interacting with the Mg+2 binding site on the polymerase; and 5) to examine the biological consequences of DNA replication across an oxidized DNA lesion (8-oxo dG) in the presence of Cr(III) or Ni(II) using a site modified bacteriophage DNA template. The primary approach will be to study the effect of these metal ions on the kinetics of DNA polymerase function in vitro and to use the results to evaluate the polymerase mechanism. O6-methyl guanine was chosen as a model DNA lesion because it can basepair with both dT and dC with different efficiencies and because it acts as a replication block for some, but not all, polymerases. Two eukaryotic and two prokaryotic DNA polymerases will be used: calf thymus DNA polymerase alpha, cloned human polymerase beta, T7 polymerase and exonuclease free T7 (Sequenase). 8-Oxo dG was chosen to look at biological consequences because chromium and nickel both increase the amount of 8-oxo dG in cellular DNA and because 8-oxo dG is weakly mutagenic in the absence of added metals. Do Ni(II) or Cr(III) increase the mutagenic potential of 8-oxo dG by increasing the mutagenic bypass of this lesion? The results of these investigations will increase our fundamental knowledge of the mechanisms of carcinogenesis of an important class of environmental agents and will provide new information on the mechanisms by which DNA polymerases achieve their high degree of replication fidelity.
| Singh, J; Snow, E T (1998) Chromium(III) decreases the fidelity of human DNA polymerase beta. Biochemistry 37:9371-8 |
| Chin, Y E; Snow, E T; Christie, N T (1994) A single stranded DNA binding protein isolated from HeLa cells facilitates Ni2+ activation of DNA polymerases in vitro. Biochemistry 33:15141-8 |
