OXYGEN RADICAL-INDUCED MUTAGENESIS IS DNA-POLYMERASE SPECIFIC

Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZα forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase α or β, transfected into E. coli, and screened for mutations by reduced α-complementation of β-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 μM CuCl, 10 μM H2O2 and 100 μM ascorbic acid results in a 3·3-fold and a 3·6-fold elevation in mutation frequency for polymerases α and β, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase α, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase β, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are against specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage. © 1994 Academic Press Limited.