RESUMO
The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Genes Bacterianos , Mitomicina/farmacologia , Resposta SOS em Genética/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/efeitos da radiação , Sequência Conservada , Dano ao DNA , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Epistasia Genética/efeitos dos fármacos , Epistasia Genética/efeitos da radiação , Deleção de Genes , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/efeitos da radiação , Mutação/genética , Taxa de Mutação , Fenótipo , Regiões Promotoras Genéticas/genética , Resposta SOS em Genética/efeitos dos fármacos , Resposta SOS em Genética/efeitos da radiação , Raios UltravioletaRESUMO
Caulobacter crescentussigma(E) belongs to the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, whose members regulate gene expression in response to distinct environmental stresses. During physiological growth conditions, data indicate that sigma(E) is maintained in reduced levels due to the action of ChrR, a negative regulator of rpoE gene expression and function. However, once bacterial cells are exposed to cadmium, organic hydroperoxide, singlet oxygen or UV-A irradiation, transcription of rpoE is induced in a sigma(E)-dependent manner. Site-directed mutagenesis indicated that residue C188 in ChrR is critical for the cadmium response while residues H140 and H142 are required for the bacterial response to organic hydroperoxide, singlet oxygen and UV-A. Global transcriptional analysis showed that sigma(E) regulates genes involved in protecting cells against oxidative damages. A combination of transcriptional start site identification and promoter prediction revealed that some of these genes contain a putative sigma(E)-dependent motif in their upstream regions. Furthermore, deletion of rpoE and two sigma(E)-dependent genes (cfaS and hsp20) impairs Caulobacter survival when singlet oxygen is constantly generated in the cells.
Assuntos
Cádmio/metabolismo , Caulobacter crescentus/genética , Peróxido de Hidrogênio/metabolismo , Fator sigma/metabolismo , Oxigênio Singlete/metabolismo , Raios Ultravioleta , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Caulobacter crescentus/efeitos da radiação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , RNA Bacteriano/genética , Transcrição GênicaRESUMO
DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.