Mol Gen Genet (1993) 239:72-76 © Springer-Verlag 1993 Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G: C T: A transversions induced by a single 8-oxoguanine residue in single-stranded DNA Masaaki Moriya, Arthur P. Grollman Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA Received: 3 September 1992/Accepted: 23 December 1992 Abstract. Oxidative damage to guanine in DNA results in the formation of 8-oxoguanine, which has been shown to induce G ~ T transversions targeted to this site. The mu- tagenicity of this lesion was studied in several mutator strains of Escherichia coli, using single-stranded DNA containing a single 8-oxoguanine residue. The frequen- cies of targeted G ~ T transversions increased markedly in rout Ystrains, while this mutagenic event was not affect- ed in mutM or mutS strains. Introdution of a mutM muta- tion into a mutY strain caused a somewhat higher fre- quency of G~T transversions than that in the mut Y strain and the effect of a routS mutation was marg!nal. We conclude that the mutYgene plays a crucial role in pre- venting targeted G ~ T mutations derived from misrepli- cation of the 8-oxoguanine-containing template DNA. Key words: Site-specific mutagenesis - Oxidative DNA damage - 8-Hydroxyguanine - Mutator Introduction Cellular DNA is damaged by oxygen-free radicals generated during endogenous oxidative processes or by exposure to oxygen radical-forming agents (Saul et al. 1987; Ames 1989). Oxidative damage to DNA is thought to play a significant role in spontaneous mutagenesis, cancer and the aging process (Ames 1983, 1989; Ames and Gold 1991). 8-Oxoguanine (8-oxoG) is created by oxidative damage to DNA and is found in significant quantities in cellular DNA (Ames and Gold 1991; Kasai and Nishimura 1991). In vitro primer extension studies showed that dCMP or dAMP is incorporated opposite 8-oxoG; dAMP is preferentially inserted by "replicative" DNA poly- merases (a and 6) and dCMP by "repair" polymerases Communicated by M. Sekiguchi Correspondence to: M. Moriya (I and 13) (Shibutani et al. 1991). In vivo studies using a single 8-oxoG residue embedded in single-stranded (ss) DNA revealed this lesion to be weakly mutagenic, induc- ing targeted G~T transversions in Escherichia coli (Wood et al. 1990; Moriya et al. 1991 ; Cheng et al. 1992) and simian kidney cells (Moriya 1993). Michaels et al. (1992) found that E. coli MutY protein, an adenine DNA-glycosylase (Au et al. 1989), removes adenine from an A: 8-oxoG pair in vitro, suggesting that the MutY protein removes adenine misincorporated opposite 8-oxoG residues after DNA replication is completed. Ac- cordingly, the weak in vivo mutagenicity of 8-oxoG em- bedded in ssDNA may be ascribed to the activity of MutY protein. In this paper, we show that 8-oxoG in ssDNA is strongly mutagenic in mutY strains of E. coli. We also report effects of other mutator genes on the mutagenicity of this lesion. Materials and methods E. coli strains used in this study were ABl157 [thr-1, ara-14, leuB6, A(gpt-proA)62, lacY1, tsx-33, SupE44, 9alK2, hisG4, rfbD1, rpsL31, kdgK51, xyl-5, mtl-1, aroE3, thi-1, rac, )q, AB 1157-Y 11 (as AB 1157 but mut IT, zgd:.'TnlO), RK1517 (as ABl157 but mutS::Tn5), RK1517-Y33 (as RK1517 but mutY, nupG511::TnlO) (Au et al. 1988), TT101 (as CC104 but mutM: :mini-tet) (Michaels et al. 1991), TT101-mutY (as TT101 but mutY: :mini-kan) and TT108 (as CC104 but mutY: :mini- tet). CC104 is ara, A(gpt-lac)5, [F'lacI378, lacZ461, proA+B +] (Cupples and Miller 1989). ABl157 was ob- tained from B. Bachmann (Yale University, New Haven, Conn.); the derivatives of CC104 were from M. Michaels and J. Miller (UCLA, Los Angeles, Calif.); and the others were from M. Modrich (Duke University, Dur- ham, N.C.). Construction of a circular ss phagemid vector con- taining a single 8-oxoG at a specific site [hereafter called ss pMS2(8-oxoG) and ss pMS2 (G) for control] and the general procedure used in this study is shown schemati-