Different repair of O6-methylguanine occurring in DNA modified by MMS in vivo or in vitro

Abstract

Lack of the adaptive response effect on the level of GC→AT transitions induced by methyl methanesulfonate (MMS) in E. coli [Sledziewska-Gójska, E. (1993) The level of GC→AT transitions induced by MMS is not affected by adaptive response of Escherichia coli K12. Mutation Res., 294, 1–8.] can be explained by MMS inactivation of the ada encoded O6-methylguanine-DNA methyltransferase [Takahashi, K.Y., Kawazoe, K., Sakumi, Nakabeppu Y. and M. Sekiguchi (1988) Activation of Ada protein as a transcriptional regulator by direct alkylation with methylating agents, J. Biol. Chem., 263, 13490–13492; Śledziewska-Gójska, E. (1995) Inactivation of O6-methylguanine-DNA methyltransferase in vivo by SN2 alkylating agents, Mutation Res., 336, 61–67]. To evaluate this explanation and clarify the origin of MMS-induced GC→AT transitions, we compared the repair of DNA treated by MMS in vivo or in vitro. Replication forms of lacZ mutants of E. coli phage M13mp18 were used to analyse the effect of the adaptive response on the frequency of GC→AT transitions occurring in control and mismatch repair deficient strains. It was shown that DNA lesions, leading to GC→AT transitions, induced by MMS in vivo are not repaired in adapted E. coli cells. In contrast, induction of the adaptive response causes efficient repair of these DNA lesions induced by MMS in vitro. This repair is consistent with the assumption that GC→AT transitions induced by MMS are originated by O6-methylguanine and that MMS treatment of the cells during in vivo mutagenesis interfere with the adaptation mediated repair of the lesion. In agreement with this we have shown that treatment of the adapted cultures with 5 mM MMS completely blocks repair of in vitro modified DNA. Increased level of GC→AT transitions induced by MMS occurs in mutS− strains. These mutations are avoided in adapted mutS- cells, when induced by MMS in vitro. This confirms that mismatch repair system of E. coli recognises mismatches formed in DNA by O6-methylguanine.