Characterization of POLQ's Function in Replication Rescue

POLQ 在复制救援中的功能特征

• 批准号: 10311939
• 负责人: Susanna Jane Stroik
• 金额: $ 7.05万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311939
• 关键词: Acute; Camptothecin; Cancerous; Cell Cycle Checkpoint; Cell Cycle Checkpoint Genes; Cell Cycle Stage; Cell Line; Cell division; Cells; Cicatrix; Comb animal structure; DNA; DNA Damage; DNA Double Strand Break; DNA Repair Pathway; DNA Replication Damage; DNA biosynthesis; DNA replication fork; Development; Engineering; Enzymes; Gatekeeping; Genes; Genome; Genomic Instability; Genomics; Goals; Individual; Kinetics; Left; Link; Malignant Neoplasms; Measures; Mediating; Microscopy; Mitosis; Molecular; Monitor; Mutate; Mutation; Neoplasm Metastasis; Nonhomologous DNA End Joining; Null Lymphocytes; Optics; Outcome; PALB2 gene; Pathway interactions; Pharmaceutical Preparations; Play; Poison; Polymerase; Proteins; Radiation therapy; Recovery; Reporter; Resistance; Resolution; Role; S Phase; Sequence Homologs; Site; Source; Speed; System; Techniques; Therapeutic; Therapeutic Intervention; Time; Tumor Suppressor Genes; Tumor Suppressor Proteins; Type I DNA Topoisomerases; Work; brca gene; cancer cell; cancer diagnosis; cancer genome; cancer prevention; cancer therapy; cell injury; chemotherapeutic agent; deep sequencing; ds-DNA; genetically modified cells; genome sequencing; genome-wide; homologous recombination; individualized medicine; insight; malignant breast neoplasm; novel; reconstruction; recruit; repaired; replication stress; response; single molecule; standard of care; targeted treatment; tumor; tumorigenesis; whole genome

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.

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不再“按规则发挥作用”的癌细胞。这是通过 引入抑制肿瘤和细胞周期检查点基因中的关键突变，该基因允许癌细胞 抵抗许多治疗性干预措施。值得注意的是，许多癌症的护理标准是用DNA治疗 破坏剂 - 化学治疗和放射疗法 - 目的是损害癌细胞以外的癌细胞 维修。这是理解在这些条件下修复DNA损伤的机制 对于预防癌症及其治疗而言，至关重要。在DNA损伤中，最严重的威胁 基因组是DNA双链断裂（DSB）。 DSB具有3种主要的维修机制 部署是为了响应它们的积累 - 同源重组（HR），经典的非理论端 - 加入，theta介导的末端加入（TMEJ）。后者在许多癌症中进行了更新，特别是 HR基因突变（BRCA1/2，PALB2等）。但是，对于这种途径扮演的角色知之甚少 在DNA复制过程中发生的休息时间，许多癌症治疗的细胞周期阶段 诱导DNA损伤。我建议研究tmej，polq中的中心蛋白在DNA中发挥作用 复制和这种作用的基因组后果。在我的初步工作中，我已经确定了Polq 缺乏使细胞对急性和长时间的cAmptothecin（CPT）治疗敏感（拓扑异构酶） 我有毒会导致复制叉倒塌。此外，Polq-Null细胞显示出高水平的终极失速 CPT暴露后的复制叉，这意味着POLQ能够修复叉子以重新启动复制。在这个 提案，我旨在研究POLQ如何促进复制叉进展，保护和恢复 诱导复制断裂。此外，我设计了一个可诱导的损坏复制叉记者系统， 我可以通过它来测量单个复制DSB的动力学和修复特征。使用这些 实验方法，我可以剖析POLQ介导的修复的分子要求和结果 破裂的叉子。最后，我试图阐明在两种HR的DNA复制过程中POLQ/TMEJ扮演的角色 以及不足的癌症，可以为癌症治疗和肿瘤动态提供机械洞察力。 基因组复制对于癌细胞分裂至关重要，因此定义了POLQ如何修复复制性断裂 促进随后的复制至关重要。