Regulation of replication and recombination intermediates

复制和重组中间体的调控

• 批准号: 10406889
• 负责人: Xiaolan Zhao
• 金额: $ 35.92万
• 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/10406889
• 关键词: Affect; Anaphase; Binding; Biochemical; Biological Assay; Cells; ChIP-seq; Complex; Cruciform DNA; DNA; DNA Damage; DNA Maintenance; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Disease; Ensure; Enzymes; Event; Excision; Face; Failure; Genetic Nondisjunction; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; In Vitro; Investigation; Lead; Link; Maintenance; Malignant Neoplasms; Mediating; Mitosis; Modeling; Molecular Genetics; Mutation; Outcome; Pathway interactions; Prevention; Process; Protein Biosynthesis; Proteins; RNA chemical synthesis; Regulation; Research; Resolution; Ribosomal DNA; Ribosomal RNA; Role; Saccharomycetales; Site; Specificity; Stress; Structure; Sumoylation Pathway; Syndrome; System; Testing; Topoisomerase; Travel; Work; Yeasts; base; cofactor; coping; diagnostic strategy; genetic regulatory protein; homologous recombination; human disease; in vivo; insight; non-histone protein; novel diagnostics; physical property; prevent; recombinational repair; recruit; repaired; treatment strategy

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 tumorigensis. 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 in yeast 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复制叉的调节， 堵塞。未能协助停滞的复制叉可导致不完整的复制和许多类型的遗传缺陷。 DNA脆性综合征和肿瘤发生的潜在改变。与DNA紧密结合的非组蛋白 （蛋白质屏障）是叉阻断的主要原因，其中很大一部分位于重复的 核糖体DNA（rDNA）。rDNA组织核仁并构成跨物种基因组的10-30%。因此，在本发明中， rDNA复制影响整体基因组稳定性以及RNA和蛋白质合成。rDNA蛋白屏障 具有独特的特征，例如由于高水平的rRNA转录和需要更大的拓扑应力， 复制体的长期维持。可以确保rDNA复制完成的机制， 挑战尚不明确。令人兴奋的是，我们最近在酵母中的数据表明，保守的八亚基Smc 5/6 复合体为应对rDNA的独特挑战提供了综合解决方案。我们发现Smc 5/6是 这对于完成rDNA的复制而不是非rDNA区域的复制是必需的。我们进一步确定Smc 5/6 限制rDNA蛋白质屏障处的复制叉逆转。我们的新数据让我们提出Smc 5/6使用 其亚基的组合活性调节rDNA蛋白质屏障处的停滞叉，并确保适当的rDNA 复制终止。我们计划使用分子、遗传和基因的组合来检验这一中心假设。 目标1中的生物化学方法。 当停滞的复制叉无法恢复时，可以修复塌陷的复制叉和未复制的DNA缺口 通过同源重组，产生重组中间体，如霍利迪连接。及时 解决这些结构对于防止有丝分裂过程中DNA缠结至关重要，这可能导致分裂后期 桥、微核形成和基因组不稳定性。我们和其他人的研究揭示了多种 调节因素是关键的霍利迪交界处删除。然而，其功能机制仍然是 被阐明。我们目前对保守的调节因子之一Esc 2蛋白的研究， 基因组稳定性，导致其功能机制的新模型。特别是，我们建议Esc 2使用 增强HJ溶解的双峰策略，包括结构贡献和SUMO介导的 机制在目标2中，我们计划测试此模型并定义Esc 2如何启用HJ清除。完成 在这个建议的目标，我们将使用高分辨率的检测在高效酵母系统。成果 这项工作将扩大我们对几个过程的认识，包括rDNA复制完成是如何在细胞内完成的。 实现，复制叉如何以特定于上下文的方式进行调节，以及重组中间体 可通过调节蛋白来辅助去除。由于这些过程与DNA损伤密切相关， 综合征和癌症，我们的研究将告知这些疾病的潜在机制，并有助于发展 新的诊断和治疗策略。