CAREER: Recovery of Replication Following DNA Damage in E.coli

在存在DNA损伤的情况下，不准确的复制是在异常分裂细胞中观察到的大多数细胞重排和突变的原因。DNA损伤，如紫外线照射引起的DNA损伤，严重损害复制基因组模板的能力，并导致复制机制的停滞和新复制DNA的间隙。本研究的总体目标是确定在复制过程中遇到的病变在体内处理和修复的细胞机制。该项目利用两种先前建立的细胞分析来监测发生在体内DNA损伤的复制叉上的分子事件。这种方法将确定遗传需求和条件，这些条件决定了DNA修复或翻译合成何时在被阻止的复制叉上起作用以恢复DNA合成。第二种方法将描述基因和机制，这些基因和机制处理和修复在被紫外线照射的细胞中新复制的DNA中观察到的缺口底物中的损伤。通过了解在DNA损伤的情况下忠实复制是如何维持的，可以阐明导致突变、基因组重排或细胞致死的条件和事件。此外，这个项目将通过加强本科课程，为学生创建一个新的分子遗传学实验室，并增加学生在这所大学的研究参与的过程来完成。该项目将显著增加参与研究的潜在青年科学家的数量，并提高该国该地区学生接触到的科学质量。

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis that are observed in abnormally dividing cells. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to copy the genomic template and leads to the arrest of the replication machinery and gaps in the newly replicated DNA. The overall objective of this research is to identify the cellular mechanism(s) by which lesions encountered during replication are processed and repaired in vivo. This project utilizes two previously established cellular assays to monitor the molecular events that occur at replication forks arrested by DNA damage in vivo. This approach will identify the genetic requirements and conditions that determine when DNA repair or translesion synthesis functions at arrested replication forks to restore DNA synthesis. A second approach will characterize the genes and mechanisms that operate to process and repair lesions in the gapped substrates that are observed to arise in the newly replicated DNA of UV-irradiated cells. Through an understanding of how faithful replication is maintained in the presence of DNA damage, the conditions and events that can lead to mutagenesis, genomic rearrangements, or cell lethality will be elucidated. In addition, this project will be accomplished through a process that strengthens the undergraduate curriculum, creates a new molecular genetics laboratory for students, and increases student participation in research at this university. This project will significantly increase the number of potential young scientists that participate in research and increase the quality of science that students are exposed to in this region of the country.