Regulation of Replication and Recombination Intermediates

复制和重组中间体的调控

• 批准号: 10414197
• 负责人: Xiaolan Zhao
• 金额: $ 7.53万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414197
• 关键词: Address; Affect; Anaphase; Binding; Biochemical; Biochemical Genetics; Biological Assay; Cells; ChIP-seq; Complex; Cruciform DNA; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Defect; Development; Diagnosis; Disease; Ensure; Excision; Failure; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Human; In Vitro; Lead; Link; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mitosis; Modeling; Molecular Genetics; Mutate; Mutation; Outcome; Process; Protein Biosynthesis; Proteins; RNA chemical synthesis; Regulation; Replicon; Research; Resolution; Ribosomal DNA; Ribosomal RNA; Role; Site; Stress; Structure; Syndrome; System; Testing; Time; Topoisomerase; Work; Yeasts; biological adaptation to stress; coping; crosslink; fitness; genetic regulatory protein; genetic testing; homologous recombination; human disease; insight; mutant; non-histone protein; novel diagnostics; overexpression; parent grant; prevent; repaired; tool; treatment strategy; tumorigenesis

Faithful duplication of the genome requires regulation of replication forks that stall at numerous template blockages. Failure to assist stalled replication forks can lead to incomplete replication and many types of genetic alterations underlying DNA fragility syndromes and tumorigenesis. Non-histone proteins tightly bound to DNA (protein barriers) are a major cause of fork blockade, and a large portion of these are located inside the repetitive ribosomal DNA (rDNA). rDNA organizes nucleoli and constitutes 10-30% of the genome across species. As such, rDNA replication influences overall genomic stability as well as RNA and protein synthesis. rDNA protein barriers have unique features such as greater topological stress due to high levels of rRNA transcription and requirement of extended maintenance of the replisome. Mechanisms that can ensure rDNA replication completion given these challenges are unclear. Excitingly, our recent data suggest that the conserved eight-subunit Smc5/6 complex provides an integrated solution for coping with unique challenges at rDNA. We found that Smc5/6 is essential for completing replication at rDNA but not at non-rDNA regions. We further determined that Smc5/6 limits replication fork reversal at rDNA protein barriers. Our new data let us propose that Smc5/6 uses the combined activities of its subunits to regulate stalled forks at rDNA protein barriers and ensure proper rDNA replication termination. We plan to test this central hypothesis using a combination of molecular, genetic, and biochemical approaches in Aim 1. When stalled replication forks fail to recover, collapsed forks and unreplicated DNA gaps can be repaired by homologous recombination, generating recombination intermediates such as Holliday junctions. Promptly resolving these structures is critical for preventing DNA entanglement during mitosis, which can lead to anaphase bridges, micronuclei formation, and genomic instability. Studies from us and others have uncovered multiple regulatory factors that are critical for Holliday junction removal. However, their functional mechanisms remain to be elucidated. Our current research on one of the conserved regulatory factors, the Esc2 protein, which is critical for genomic stability, leads to new models for its functional mechanisms. In particular, we suggest that Esc2 uses a bimodal strategy for enhancing HJ dissolution, including both a structural contribution and a SUMO-mediated mechanism. In Aim 2, we plan to test this model and define how HJ clearance is enabled by Esc2. To accomplish the goals in this proposal, we will use high-resolution assays in the highly effective yeast system. Outcomes of this proposed work will expand our view of several processes, including how rDNA replication completion is achieved, how replication fork is regulated in a context-specific manner, and how recombination intermediate removal can be assisted by regulatory proteins. As these processes are intimately linked to DNA damage syndromes and cancers, our studies will inform mechanisms underlying these diseases, and help to develop new diagnostic and treatment strategies.

基因组的忠实复制需要调节复制叉子，而复制叉子在众多模板上停滞不前 堵住了。未能协助停滞的复制分叉可能会导致复制不完整和多种类型的 DNA脆性综合征和肿瘤发生的基因改变。紧密结合的非组蛋白 TO DNA(蛋白质屏障)是叉子堵塞的主要原因，其中很大一部分位于 重复核糖体DNA(RDNA)。RDNA组织核仁，占整个基因组的10%-30% 物种。因此，rDNA复制影响整个基因组的稳定性以及RNA和蛋白质的合成。 RDNA蛋白屏障具有独特的特征，例如由于高水平的rRNA而产生更大的拓扑应力 复制体的转录和延长维持的要求。可以确保rDNA的机制 鉴于这些挑战，复制完成情况尚不清楚。令人兴奋的是，我们最近的数据表明 保守的八亚基Smc5/6复合体为应对独特的挑战提供了一个集成的解决方案 RDNA。我们发现Smc5/6是在rDNA区域完成复制所必需的，但在非rDNA区域不是。我们 进一步确定Smc5/6在rDNA蛋白屏障上限制了复制分叉的逆转。我们的新数据让我们 建议Smc5/6利用其亚基的联合活性来调节rDNA蛋白的停滞分叉 障碍并确保适当的rDNA复制终止。我们计划使用一个 在目标1中结合了分子、遗传和生化方法。 当停止的复制分叉无法恢复时，折叠的分叉和未复制的DNA缺口可能会 通过同源重组修复，产生重组中间体，如Holliday连接。 及时解决这些结构对于防止有丝分裂过程中的DNA纠缠至关重要，这可能会导致 到后期桥、微核形成和基因组不稳定。我们和其他人的研究已经 发现了对Holliday连接移除至关重要的多个调节因素。然而，它们的功能 其作用机制尚待阐明。我们目前对保守调控因子之一的研究 ESc2蛋白对基因组的稳定性至关重要，为其功能机制提供了新的模型。在……里面 特别是，我们建议ESc2使用双峰策略来促进HJ的溶解，包括 结构性贡献和相扑中介机制。在目标2中，我们计划测试该模型并定义 HJ净空由ESc2启用。为了实现本提案中的目标，我们将使用高分辨率分析 在高效的酵母系统中。这项拟议工作的成果将扩大我们对以下几个问题的看法 过程，包括如何完成rDNA复制、如何在 特定于上下文的方式，以及调控蛋白如何帮助重组中间产物的去除。 由于这些过程与DNA损伤综合症和癌症密切相关，我们的研究将告知 这些疾病的潜在机制，并有助于制定新的诊断和治疗战略。