J. Mol. Biol. (1994) 235, 33-41 Oxygen Radical Induced Mutagenesis is DNA Polymerase Specific Daniel I. Feig ~ and Lawrence A. Loeb2t Joseph Gottstein Memorial Cancer Research Laboratory Departments of 1Biochemistry and 2pathology University of Washington Seattle, WA 98195, U.S.A. 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 lacZa forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase a or fl, transfected into E. coli, and screened for mutations by reduced a-complementation of fl-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/~M CuCl, 10/~M H202 and 100 #M ascorbic acid results in a 3"3-fold and a 3"6-fold elevation in mutation frequency for polymerases a and fl, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase a, 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 fl, 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 again 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. Keywords: polymerase a; polymerase fl; reactive oxygen species; DNA damage; mutation At a fundamental level the source of many mutations is misincorporation by DNA polymer- ases. Misincorporation can result from the infidelity of DNA polymerases or from altered base-pairing properties secondary to unrepaired DNA damage such as that resulting from oxygen free radicals. Ames and colleagues estimate that the generation of reactive oxygen species by normal cellular processes results in as many as 10,000 DNA damaging events per eucaryotic cell per day (Fraga et al., 1990). Despite the cellular mechanisms for the repair of this damage, it is likely that a fraction of these lesions escape repair and cause mutations. We have investigated the consequences of unrepaired oxygen species damage by copying oxidized DNA with puri- fied mammalian DNA polymerases. t Author to whom all correspondence should be addressed. 0022-2836/94/010033-09 $08.00/0 The four eucaryotic, nuclear DNA polymerases are a study in contrasts. Mammalian polymerases a, 5 and ~ are involved in DNA replication and their cellular levels increase during the transition of non- dividing to actively replicating cells (Linn, 1991; Wang, 1991). By contrast, pol-fl$ is expressed at the same levels in resting and in replicating cells (Zmudzka et al., 1988) although it is inducible by some types of DNA damage (Fornace et al., 1989). The replicative polymerases have large catalytic subunits, 125 to 215 kDa, and are intimately asso- ciated with additional subunits of varying function while fl is isolated as a single 38 kDa polypeptide. 33 :~Abbreviations used: pol-fl, recombinant rat DNA polymerase fl; pol-~, calf thymus DNA polymerase ~-primase; SOD, superoxide dismuta~e; 8-OHdGuo, 8-hydroxy-2'-deoxyguanosine; me~Guo, O-6-methyl-2'-deoxyguanosine. © 1994 AcademicPress Limited