Analysis of global gene expression and double-strand-break formation in DNA adenine methyltransferase- and mismatch repair-deficient Escherichia coli.
DNA adenine methylation by DNA adenine methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.
Robbins-Manke
Jennifer L
JL
Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Ave., 56-670, Cambridge, MA 02139, USA.
Zdraveski
Zoran Z
ZZ
Marinus
Martin
M
Essigmann
John M
JM
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D011995:Recombination, Genetic / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004247:DNA; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli