Characterization of POLQ's Function in Replication Rescue

项目摘要 基因组不稳定是已知的癌症的一个特征，它会在细胞分裂过程中产生基因变化。仅此一项 可以推动肿瘤的形成，并进化出不再“按规则行事”的癌细胞。这可以通过以下方式实现 肿瘤抑制基因和细胞周期检查点基因的关键突变允许癌细胞 抵制许多治疗干预。值得注意的是，许多癌症的治疗标准是用dna治疗。 破坏性药物--化学疗法和放射疗法--其目标是破坏癌细胞 修理。因此，了解在这些条件下修复DNA损伤的机制是 对于癌症的预防和治疗都是最重要的。在DNA损伤中，最严重的威胁 对基因组来说是DNA双链断裂(DSB)。DSB有3种主要的修复机制，可以是 部署以响应它们的积累-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)。后者在许多癌症中表达上调，特别是在 哪些HR基因发生突变(BRCA1/2、PALB2等)。然而，人们对这一途径所起的作用知之甚少。 在修复DNA复制过程中发生的断裂时，许多癌症治疗药物在细胞周期阶段 导致DNA损伤。我建议研究TMEJ中的中心蛋白POLQ在DNA中所起的确切作用 复制和这种角色的基因组后果。在我的初步工作中，我已经确立了POLQ 缺乏使细胞对喜树碱(CPT)的急性和长期治疗敏感，喜树碱是一种拓扑异构酶 I毒药会导致复制叉子坍塌。此外，POLQ-NULL单元显示高水平的终端停滞 CPT暴露后的复制分叉，这意味着POLQ能够修复复制重启的分叉。在这 提案，我的目标是研究POLQ如何有助于复制分叉的进展、保护和在复制后的恢复 复制中断的诱导。此外，我还设计了一种可诱导的断裂复制叉状报告系统， 我可以用它来测量动力学和修复单个复制双链断裂的特征。使用这些 实验方法，我可以剖析POLQ介导的修复的分子需求和结果 折断的叉子。最后，我试图阐明POLQ/TMEJ在两个HR熟练者的DNA复制过程中所起的作用 和缺乏癌症，这将提供对癌症治疗和肿瘤动力学的机械性洞察。 基因组复制对癌细胞分裂至关重要，因此决定了POLQ修复复制断裂的方式 并且便于后续复制是必不可少的。

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.