Replication fork dynamics and repair by Rad51 paralogues after DNA alkylation

DNA 烷基化后 Rad51 旁系同源物的复制叉动力学和修复

• 批准号: 10736647
• 负责人: Kara A Bernstein
• 金额: $ 33.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10736647
• 关键词: Replication; fork; dynamics; repair; Rad51

7. Project Summary Misrepair of DNA damage is a hallmark of cancer. We discovered that the budding yeast Shu complex is a conserved regulator of DNA repair through a central role in Rad51 regulation. Rad51 functions during the high fidelity homologous recombination pathway to find and invade a homologous template for repair and also during replication fork protection and restart. Rad51 is tightly regulated in cells by accessory proteins, collectively called the Rad51 mediators, including the Shu complex. In humans, misregulation of hRAD51 or its mediators is associated with cancer predisposition (particularly breast and ovarian cancers) and Fanconi anemia, which is also characterized by anemia and cancer. We found that disruption of the yeast Shu complex leads to cellular death specifically upon exposure to alkylation induced DNA damage. Alkylation damage is caused by a myriad of industrial and consumer-based sources and is pervasive in our environment. DNA alkylation leads to replication stress and DNA damage. If DNA is alkylated during replication, then the replication fork can stall or collapse, and many repair mechanisms can be utilized to tolerate, bypass, or repair the damaged DNA. How a cell commits to a specific repair pathway is largely known. In budding yeast, the Shu complex is critical in the processing of replication forks damaged by alkylating agents. This complex is highly conserved throughout eukaryotes and contains the Rad51 paralogs, proteins that are structurally similar to the central DNA repair protein Rad51 and are mutated in cancer. In this study, we aim to elucidate the role of the yeast and human Shu complexes in repair of DNA alkylation damage at a replication fork. We are testing the hypothesis that the Shu complex is a critical key regulator of DNA damage tolerance at a replication fork by specifically recognizing alkylation induced DNA damage to promote Rad51-mediated template switch and protect forks from double-strand break induction by AP endonucleases. Using what we learn in yeast to quickly and efficiently identify key substrates, residues, and protein targets, we will expand our studies into human cell lines where we will investigate the role of the human Shu complex in tolerance of alkylation damage. In addition, we will identify at risk individuals harboring mutations in these important genes that may be more sensitive to DNA alkylation damage and therefore susceptible to cancer. Collectively, these studies will provide key insights into the role of the Shu complex in tolerance of DNA alkylation damage and elucidate how this complex promotes error-free DNA repair to prevent genetic instability and cancer. !

7。项目摘要 DNA损伤的虚假是癌症的标志。我们发现发芽的酵母菌综合体是 通过在RAD51调节中的中心作用的DNA修复的保守调节剂。 RAD51在高中功能 保真性同源重组途径，以查找和入侵一个同源模板进行维修，还 在复制叉保护和重新启动过程中。 RAD51受辅助蛋白在细胞中的严格调节， 共同称为RAD51介体，包括SHU综合体。在人类中，HRAD51或 它的介体与癌症的易感性（尤其是乳腺癌和卵巢癌）和Fanconi有关 贫血，也以贫血和癌症为特征。我们发现酵母菌复合物的破坏 暴露于烷基化引起的DNA损伤时，会导致细胞死亡。烷基化损伤是 由无数的工业和消费者来源引起的，在我们的环境中普遍存在。脱氧核糖核酸 烷基化导致复制应力和DNA损伤。如果在复制过程中DNA被烷基化，则 复制叉可以停滞或崩溃，许多维修机制可用于耐受，绕过或维修 受损的DNA。细胞如何遵守特定的修复途径是广为人知的。在萌芽的酵母中 SHU复合物对于处理烷基化剂损坏的复制叉的处理至关重要。这个复合物是 在整个真核生物中高度保守，并包含Rad51旁系同源物，结构相似的蛋白质 到中央DNA修复蛋白RAD51，并在癌症中突变。在这项研究中，我们旨在阐明角色 在复制叉上修复DNA烷基化损伤的酵母和人类SHU复合物的。我们是 测试SHU复合物是复制时DNA损伤耐受性的关键关键调节剂的假设 通过特异性识别烷基化诱导的DNA损伤以促进RAD51介导的模板开关 并保护叉子免受AP核酸内切酶的双链破裂诱导。利用我们在酵母中学到的东西 快速有效地识别关键底物，残基和蛋白质靶标，我们将把研究扩展到 人类细胞系，我们将研究人类SHU复合物在烷基化耐受性中的作用 损害。此外，我们将确定在这些重要基因中具有突变的风险个体，这些基因可能 对DNA烷基化损伤更加敏感，因此易受癌症。总的来说，这些研究 将提供有关SHU复合物在DNA烷基化损伤耐受性和阐明中的作用的关键见解 该复合物如何促进无错误的DNA修复，以防止遗传不稳定性和癌症。 呢