Mechanisms of mistranslation-mediated mutator response

描述（由申请人提供）：突变在癌症起始和遗传疾病，以及微生物发病机制和药物耐药性中发挥关键作用。由于突变也会产生进化所需的遗传变异，细胞不仅具有在正常生长条件下避免过度突变的机制，而且还具有促进突变以响应环境和生理压力的机制。在这个提议中，我们将研究最近发现的突变途径，该途径被发现是由编码tRNA的基因中的突变（mutA）触发的。我们在之前资助期的研究有几个重要发现：由蛋白质合成中的遗传缺陷引起的误翻译，或暴露于抗生素链霉素，可以诱导这种诱变途径，称为TSM（翻译应激诱导诱变）。TSM不同于已知的SOS诱变途径，在一定条件下似乎需要同源重组功能。TSM似乎是由DMA聚合酶III的一种修饰形式介导的易出错的DMA合成。这些发现暗示了翻译和DMA复制之间存在先前未描述的联系，并提出了一种可能性，即对细胞存活、适应、癌症起始和衰老具有重要意义的突变表型可能是通过比以前认识到的更大的靶基因库中的缺陷产生的。抗生素暴露和氨基酸饥饿对这一途径的诱导表明，环境和生理应激也可以通过这一途径增加诱变。我们的工作进一步表明，氨基糖苷类抗生素通过这种以前未被认识到的机制具有致突变风险。在这里，我们建议通过以下三个特定目的来研究误翻译导致这种突变表型的机制。(1)验证直接误译复制性DMA聚合酶导致TSM表型的假设。(2)验证在Rec- mutA细胞中存在选择性杀伤经历偶发性异常复制周期的细胞的假设。(3)验证TSM表型是由偶发性超突变周期引起的假说。

DESCRIPTION (provided by applicant): Mutations play critical roles in cancer initiation and genetic disorders, as well as in microbial pathogenesis and resistance to drugs. Because mutations also generate genetic variability that is required for evolution, cells have mechanisms not only for avoiding excess mutation during normal growth conditions, but also for promoting mutations in response to environmental and physiological stress. In this proposal we will investigate of a recently discovered pathway for mutagenesis that was found to be triggered, surprisingly, by a mutation (mutA) in a gene coding for a tRNA. Our investigation in the previous funding period has led to several important discoveries: mistranslation induced by genetic defects in protein synthesis, or exposure to the antibiotic streptomycin, can induce this mutagenic pathway, named TSM for translational stress-induced mutagenesis. TSM is distinct from the well known SOS mutagenesis pathway, and appears to require homologous recombination functions under certain conditions. TSM appears to be mediated by error-prone DMA synthesis by a modified form of DMA polymerase III. These findings imply the existence of previously undescribed links between translation and DMA replication, and raise the possibility that mutator phenotypes with significance for cell survival, adaptation, cancer initiation, and aging can arise through defects in a much larger repertoire of target genes than previously recognized. The induction of this pathway by antibiotic exposure, and potentially by amino acid starvation suggest that environmental and physiological stress can also increase mutagenesis by this pathway. Our work further shows that aminoglycoside antibiotics pose a mutagenic risk through this previously unrecognized mechanism. Here, we propose to investigate the mechanisms by which mistranslation leads this mutator phenotype through the following three Specific Aims. (1) Test the hypothesis that direct mistranslation of a replicative DMA polymerase leads to the TSM phenotype. (2) Test the hypothesis that in Rec- mutA cells there is selective killing of cells undergoing episodic aberrant replication cycles. (3) Test the hypothesis that the TSM phenotype results from episodic hyper-mutagenesis cycles.