RESUMEN
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.
Asunto(s)
Reparación del ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutación , Recombinación Genética/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Regulación hacia Arriba/genéticaRESUMEN
The sensitivity of a panel of DNA repair-defective bacterial strains to BLM was investigated. Escherichia coli recA cells were far more sensitive than were uvrA, dam-3, and mutM mutY strains, underscoring the importance of RecA to survival. Strains recBCD and recN, which lack proteins required for double strand break (DSB) repair, were highly sensitive to BLM, while recF cells were not. The requirement for DSB-specific enzymes supports the hypothesis that DSBs are the primary cause of bleomycin cytotoxicity. The acute sensitivity of recN cells was comparable to that of recA, implying a central role for the RecN protein in BLM lesion repair. The Holliday junction processing enzymes RecG and RuvC were both required for BLM survival. The recG ruvC double mutant was no more sensitive than either mutation alone, suggesting that both enzymes participate in the same pathway. Surprisingly, ruvAB cells were no more sensitive than wildtype, implying that RuvC is able to perform its role without RuvAB. This observation contrasts with current models of recombination in which RuvA, B, and C function as a single complex. The most straightforward explanation of these results is that DSB repair involves a structure that serves as a good substrate for RecG, and not RuvAB.
Asunto(s)
Proteínas Bacterianas/fisiología , Bleomicina/toxicidad , Enzimas de Restricción del ADN/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de los fármacos , Mutágenos/toxicidad , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Recombinación GenéticaRESUMEN
Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.
