RESUMO
UV-induced mutagenesis is, to greater extent, a phenomenon dependent on translesion synthesis (TLS) and regulated by the SOS response in bacteria. Caulobacter crescentus, like many bacterial species, employs the ImuABC (ImuAB DnaE2) pathway in TLS. To have a better understanding of the characteristics of UV-induced mutagenesis in this organism, we performed a whole genome analysis of mutations present in survivors after an acute UVC exposure (300 J/m2). We found an average of 3.2 mutations/genome in irradiated samples, distributed in a mutational spectrum consisting exclusively of base substitutions, including tandem mutations. Although limited in conclusions by the small number of mutations identified, our study points to the feasibility of using whole-genome sequencing to study mutagenesis occurring in experiments involving a single acute exposure to genotoxic agents.
Assuntos
Caulobacter crescentus , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Proteínas de Bactérias/genética , Mutagênese , Dano ao DNA/genética , Reparo do DNA/genéticaRESUMO
imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.
Assuntos
Caulobacter crescentus/metabolismo , Dano ao DNA , Replicação do DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Mutagênese , Resposta SOS em Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
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.