title: Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.
abstract: In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warszawa, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Wrzesiński
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Nieminuszczy
authors.forename: Anna
authors.initials: A
authors.lastname: Sikora
authors.forename: Damian
authors.initials: D
authors.lastname: Mielecki
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Chojnacka
authors.forename: Marek
authors.initials: M
authors.lastname: Kozłowski
authors.forename: Joanna
authors.initials: J
authors.lastname: Krwawicz
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Grzesiuk
mesh_terms: D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D008741:Methyl Methanesulfonate; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009153:Mutagens; D013014:SOS Response, Genetics; D014157:Transcription Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009153:Mutagens; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria; D001120:Arginine; D008741:Methyl Methanesulfonate; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli