Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli
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O R I GI N A L P A P E R
T. Watanabe á G. van Geldorp á T. Najrana E. Yamamura á T. Nunoshiba á K. Yamamoto
Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli Received: 29 February 2000 / Accepted: 31 August 2000 / Published online: 25 October 2000 Ó Springer-Verlag 2000
Abstract We examined whether strand identity with respect to DNA replication in¯uences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The speci®city of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that diered only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C ® T:A transversions (>64%), while the majority in the mutT strain were base substitutions (>92%), predominantly A:T ® C:G transversions (>91%). The distributions of frequently mutated sites of G:C ® T:A and A:T ® C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not dier markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis. Key words DNA replication á Leading strand á Lagging strand á 8-OxoG á 8-OxodGTP
Introduction Reactive oxygen species (ROS) such as hydroxy radicals, formed in cells as by-products of aerobic metabolism or during oxidative stress, are highly reactive and produce complex patterns of DNA modi®cations (Boiteux et al. 1992; Dizdaroglu 1992). Several lines of evidence suggest that an oxidatively damaged form of guanine in DNA, 8-oxo-7,8-dihydroguanine (8-oxoG), Communicated by R. Devoret T. Watanabe á G. van Geldorp á T. Najrana E. Yamamura á T. Nunoshiba á K. Yamamoto (&) Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan E-mail: [email protected] Tel.: +81-22-2176706; Fax: +81-22-2176706
is highly mutagenic, inducing G:C ® T:A transversion both in vitro and in vivo (Michaels and Miller 1992; Grollman and Moriya 1993). ROS also oxidizes nucleotides to form 8-oxo-7,8-dihydro-2¢-deoxyguanosine 5¢triphosphate (8-oxodGTP), which can be incorporated opposite adenine as well as cytosine in DNA (Maki and Sekiguchi 1992). In this case, both A:T ® C:G and G:C ® T:A transversions would be induced (Cheng et al. 1992). To minimize the mutagenic eects caused by oxidized guanine, Escherichia coli possesses two DNA glycosylase activities and one phosphatase activity that prevent spontaneous mutagenesis by 8-oxoG and 8-oxodGTP; the MutM protein, which excises 8-oxoG residues in DNA (Tchou et al. 1991; Michaels et al. 1992b; Yamamoto et al. 1997), the MutY protein, which excises the adenine residues incorporated opposite 8-oxoG (Au et al. 1989; Michaels et al. 1992b; Yamamoto et al. 1997) and MutT, which hydrolyzes 8-oxodGTP to 8oxodGMP (Maki and Sekiguchi 1992). Inactivation of both the mutM and the mutY genes in E. coli results in a s
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