title: E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.
abstract: Alkylating agents are known to induce the formation of O6-alkylguanine (O6-alkG) and O4-alkylthymine (O4-alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O6-methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O6-ethylguanine. However, the manner by which O4-alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O4-alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding β-galactosidase. Such AT-to-GC mutations should be induced through the formation of O4-alkT at AT base pairs. As expected, an O6-alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O6-alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O4-alkylthymine. We hypothesize that the MutS protein recognizes the O4-alkT:A base pair more efficiently than O4-alkT:G. Such a distinction would result in misincorporation of G at the O4-alkT site, followed by higher mutation frequencies in wild-type cells, which have MutS protein, compared to MMR-deficient strains.
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Nakano
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan; Nihon Pharmaceutical University, Ina, Kita-Adachi-Gun, Saitama, 362-0806, Japan.
authors.forename: Eizo
authors.initials: E
authors.lastname: Takahashi
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan.
authors.forename: Sakae
authors.initials: S
authors.lastname: Arimoto
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan.
authors.forename: Keinosuke
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: Nihon Pharmaceutical University, Ina, Kita-Adachi-Gun, Saitama, 362-0806, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Negishi
authors.affiliation: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan; Nihon Pharmaceutical University, Ina, Kita-Adachi-Gun, Saitama, 362-0806, Japan. Electronic address: tomoeisaka@gmail.com.
authors.forename: Tomoe
authors.initials: T
authors.lastname: Negishi
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D020029:Base Pairing; D053843:DNA Mismatch Repair / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation
chemical_list: D000477:Alkylating Agents
keywords: E. coli; Mismatch repair (MMR); Mutagenesis; Mutagenic repair; Nucleotide excision repair (NER); O(4)-alkylthymine; O(6)-alkylguanine-DNA alkyltransferase (AGT)