Characterization of POLQ's Function in Replication Rescue

项目摘要 基因组不稳定性是已知在细胞分裂期间产生遗传改变的癌症的标志。一个人 可以驱动肿瘤发生并进化出不再“按规则行事”的癌细胞。这是通过 在肿瘤抑制基因和细胞周期检查点基因中引入关键突变， 抵抗许多治疗干预。值得注意的是，许多癌症的护理标准是用DNA治疗， 破坏剂-化疗和放射治疗-目的是破坏癌细胞， 修复.因此，了解在这些条件下DNA损伤修复的机制是重要的。 对癌症的预防和治疗都至关重要。在DNA损伤中， 是DNA双链断裂（DSB）。DSB有3种主要的修复机制， 部署在响应其积累-同源重组（HR），经典的非同源末端- 连接和Theta介导的末端连接（TMEJ）。后者在许多癌症中上调，特别是在那些癌症中。 哪些HR基因发生突变（BRCA 1/2、PALB 2等）。然而，很少有人知道这一途径所起的作用， 在修复DNA复制过程中发生的断裂中，DNA复制是一个细胞周期阶段，许多癌症治疗药物 导致DNA损伤。我建议研究TMEJ中的中心蛋白POLQ在DNA过程中所起的确切作用， 复制和这种作用的基因组后果。在我的初步工作中，我已经建立了POLQ 缺乏使细胞对急性和长期喜树碱（CPT）治疗敏感，喜树碱是一种拓扑异构酶， I毒药会导致复制叉崩溃。此外，POLQ-无效细胞显示高水平的终末停滞， CPT暴露后复制分叉，这意味着POLQ能够修复分叉以重新启动复制。在这 建议，我的目标是研究POLQ如何有助于复制分叉的进展，保护和恢复后， 诱导复制断裂。此外，我还设计了一个可诱导的断裂复制叉报告系统， 我可以用它来测量单个复制性DSB的动力学和修复特征。使用这些 通过实验方法，我可以剖析POLQ介导的修复的分子要求和结果， 坏掉的叉子最后，我试图阐明的作用POLQ/TMEJ发挥在DNA复制过程中，在HR熟练 和缺陷型癌症，这将为癌症治疗和肿瘤动力学提供机制性见解。 基因组复制对于癌细胞分裂至关重要，因此定义了POLQ如何修复复制断裂 并便于随后的复制是必不可少的。

Project Summary Genomic instability is a hallmark of cancer known to generate genetic alterations during cell division. It alone can drive oncogenesis and evolve cancer cells that no longer “play by the rules”. This is accomplished via introduction of key mutations in tumor suppressor and cell cycle checkpoint genes that allow cancer cells to resist many therapeutic interventions. Notably, the standard of care for many cancers is treatment with DNA damaging agents – chemotherapeutics and radiation therapy – with the goal of damaging cancer cells beyond repair. Thus, understanding the mechanisms by which DNA damage is repaired under these conditions is of the upmost importance for both cancer prevention and its treatment. Amongst DNA damage, the most severe threat to the genome are DNA double strand breaks (DSBs). DSBs have 3 major repair mechanisms which can be deployed in response to their accumulation – homologous recombination (HR), classical non-homologous end- joining, and Theta-mediated end-joining (TMEJ). The latter is upregulated in many cancers, specifically those in which HR genes are mutated (BRCA1/2, PALB2, etc.). However, little is known about the role this pathway plays in repairing breaks which occur during DNA replication, a cell cycle stage in which many cancer therapeutics induce DNA damage. I propose to study the precise role the central protein in TMEJ, POLQ, plays during DNA replication and the genomic consequences of such a role. In my preliminary work, I have established that POLQ deficiency renders cells sensitive to both acute and prolonged Camptothecin (CPT) treatment, a Topoisomerase I poison which induces replication fork collapse. Further, POLQ-null cells display high levels of terminally stalled replication forks after CPT exposure, implying POLQ is capable of repairing forks for replication restart. In this proposal, I aim to study how POLQ contributes to replication fork progression, protection, and recovery after the induction of replicative breaks. Further, I have engineered an inducible broken replication fork reporter system, with which I can measure the kinetics and repair signatures of individual replicative DSBs. Using these experimental approaches, I can dissect the molecular requirements and outcomes of POLQ-mediated repair of broken forks. Finally, I seek to elucidate the role POLQ/TMEJ plays during DNA replication in both HR-proficient and deficient cancers which would provide mechanistic insight into cancer treatments and tumor dynamics. Genome duplication is essential for cancer cell division and thus defining how POLQ repairs replicative breaks and facilitates subsequent replication is essential.