Mechanisms of DNA Double-strand Break End Resection and Repair Pathway Choice

DNA双链断裂末端切除机制及修复途径选择

• 批准号: 10658092
• 负责人: Patrick Sung
• 金额: $ 50.55万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658092
• 关键词: Amino Acids; Antineoplastic Agents; Attenuated; Award; BARD1 gene; BRCA deficient; BRCA1 gene; Biochemical; Biochemistry; Biological; Cell Cycle; Cell Cycle Progression; Cell Death; Cells; Cellular biology; Chromatin; Chromosomal Breaks; Chromosomes; Complex; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA replication fork; Defect; Deoxyribonucleases; Development; Disease; Double Strand Break Repair; Drug resistance; EXO1 gene; Environment; Enzymes; Epigenetic Process; Excision; Exposure to; Failure; Filament; Foundations; Functional disorder; G1 Phase; G2 Phase; Genetic; Genomic Instability; Goals; Grant; Human; Lead; Length; Link; Malignant Neoplasms; Mediating; Neoplastic Cell Transformation; Nonhomologous DNA End Joining; Outcome; Pathway interactions; Poly(ADP-ribose) Polymerase Inhibitor; Polymerase; Procedures; Process; Proteins; Rad51 recombinase; Reader; Regulation; Resected; Resistance; Ribose; S phase; Saccharomyces cerevisiae; Saccharomycetales; Single-Stranded DNA; System; Tail; Testing; Therapeutic; Tumor Suppressor Proteins; cancer therapy; chemotherapeutic agent; combinatorial; endonuclease; helicase; in vivo; inhibitor; insight; mutant; novel; p53-binding protein 1; predictive marker; protein complex; protein purification; reconstitution; recruit; repaired; response; restoration; spleen exonuclease; targeted cancer therapy; targeted treatment; tool; treatment response; tumor

ABSTRACT DNA double strand breaks (DSBs) are induced by environmental and chemotherapeutic agents, and during encounters of the DNA replication machinery with DNA damage. The two major, mechanistically distinct DSB repair pathways are non-homologous DNA end joining (NHEJ) and homology-directed repair (HDR). NHEJ is efficient but error-prone, while HDR is inherently accurate and represents the preferred repair tool for DNA replication-associated DSBs. HDR commences with the resection of the 5’-terminated strand at break ends to generate a DNA tail that serves as the template for assembly of the RAD51 recombinase filament. DSB repair pathway choice is linked to cell cycle progression and is determined by whether or not a DSB undergoes extensive resection. Long-range resection is principally mediated by the 5’-3’ exonuclease EXO1 or the BLM helicase-DNA2 endonuclease. The chromatin reader 53BP1 nucleates the formation of a higher order ensemble that harbors the RIF1 protein and the hetero-tetrameric Shieldin complex at DSB ends to block end resection in the G1 phase of the cell cycle. The restrictive action of the 53BP1 axis is alleviated by BRCA1- BARD1 in S and G2 phases via mechanisms that are poorly understood. Thus, BRCA1-deficient tumors, on account of their HDR-deficiency, are particularly vulnerable to PARP inhibitors (PARPi) due to synthetic lethality. However, dysfunction in the 53BP1 axis leads to HDR restoration and PARPi resistance. Importantly, we now have compelling evidence that RIF1 and Shieldin strongly restrict the activity of the DNA end resection enzymes. To elucidate the underpinnings of the DNA end resection restriction circuitry, we will employ a combinatorial approach encompassing reconstitution biochemistry and cell biology to: (1) Delineate how RIF1 and Shieldin interfere with the activity of the 5’-3’ exonuclease EXO1 and of the helicase-endonuclease complex BLM-DNA2 in DNA end resection and (2) Interrogate BRCA1-BARD1 for its ability to overcome the restriction of DNA end resection imposed by RIF1 and Shieldin. Our studies will elucidate the intricate regulatory networks that control DNA end resection onset and efficiency. Our endeavors will not only illuminate the mechanistic principles of DSB repair pathway choice, but will also exert a major impact in our understanding of how failure to properly process DSBs lead to neoplastic cell transformation and cancer, and will provide actionable information to help guide the development of targeted cancer therapies to treat BRCA- deficient cancers and circumvent drug resistance.

抽象的 DNA双链断裂（DSB）是由环境和化学治疗剂诱导的，在 DNA复制机制的遇到具有DNA损伤的遇到。这两个主要的，机械不同的DSB 维修途径是非理论DNA端连接（NHEJ）和同源指导修复（HDR）。 nhej是 高效但容易出错，而HDR本质上是准确的，代表DNA的首选维修工具 复制相关的DSB。 HDR开始于休息时的5'终止链的切除 为了产生作为Rad51重组酶丝组装的模板的DNA尾巴。 DSB 修复途径选择与细胞周期的进程有关，并由DSB确定 广泛的切除。远程切除主要由5'-3'外切核酸酶Exo1或BLM介导 解旋酶-DNA2核酸内切酶。染色质读取器53BP1核能形成高阶 携带RIF1蛋白质和异光四聚体屏蔽蛋白复合物的合奏在DSB末端到达末端 在细胞周期的G1阶段切除。 BRCA1-缓解了53BP1轴的限制作用 S和G2阶段的Bard1通过知之甚少的机制。那是BRCA1缺陷肿瘤， 由于合成而引起的HDR缺乏率的说明特别容易受到PARP抑制剂（PARPI）的影响 致死性。但是，53BP1轴的功能障碍会导致HDR恢复和PARPI抗性。重要的是， 现在，我们有令人信服的证据表明RIF1和Shieldin强烈限制了DNA末端切除的活性 酶。为了阐明DNA结束对帐限制电路的基础，我们将采用一个 组合方法包括重建生物化学和细胞生物学的组合方法：（1）描述RIF1如何 和Shieldin干扰5'-3'外丝丝酶Exo1的活性和解旋酶 - 核酸酶的活性 DNA末端切除术中的复杂BLM-DNA2，（2）询问BRCA1-BARD1的能力 RIF1和SHIELDIN对DNA终端切除的限制。我们的研究将阐明复杂的 控制DNA最终切除开始和效率的调节网络。我们的努力不仅会照亮 DSB维修途径选择的机械原理，但也将对我们的 了解未能正确处理DSB如何导致肿瘤细胞转化和癌症以及 将提供可行的信息，以帮助指导靶向癌症疗法的发展以治疗BRCA- 不足的癌症和抗药性。