Regulation of Replication and Recombination Intermediates

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分叉可能会导致不完整的复制和许多类型的 DNA脆性综合征和肿瘤发生的遗传改变。紧密结合的非组蛋白 DNA（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复制影响整体基因组稳定性以及RNA和蛋白质合成。 rDNA蛋白质屏障具有独特的特征，例如由于高水平的rRNA， 转录和复制体延长维持的需要。可以确保rDNA 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Smc 5/6复合物提供了一个综合的解决方案，以应对独特的挑战， rDNA。我们发现Smc 5/6是完成rDNA复制所必需的，但不是在非rDNA区域。我们 进一步确定Smc 5/6限制了rDNA蛋白质屏障处的复制叉逆转。我们的新数据让我们 Smc 5/6是利用其亚基的联合活性来调节rDNA蛋白质上的停滞叉 屏障并确保适当的rDNA复制终止。我们计划使用一个 目标1中的分子、遗传和生物化学方法的组合。 当停滞的复制叉无法恢复时，崩溃的复制叉和未复制的DNA间隙可能会被破坏。 通过同源重组修复，产生重组中间体，如霍利迪连接。 准确地解析这些结构对于防止有丝分裂过程中的DNA缠结至关重要， 后期桥，微核形成，和基因组不稳定性。我们和其他人的研究 揭示了多个调节因素，是关键的霍利迪交界处删除。然而，其功能 机制仍有待阐明。我们目前对保守的调节因子之一， esc 2蛋白是基因组稳定性的关键蛋白，为研究其功能机制提供了新的模型。在 特别是，我们建议Esc 2使用双峰策略来增强HJ溶解，包括 结构贡献和SUMO介导的机制。在目标2中，我们计划测试这个模型，并定义如何 通过Esc 2启用HJ清除。为了实现本提案中的目标，我们将使用高分辨率分析 在高效酵母系统中。这项拟议工作的结果将扩大我们对几个问题的看法。 过程，包括如何实现rDNA复制完成，复制叉如何在一个 背景特异性方式，以及重组中间体去除如何可以通过调节蛋白来辅助。 由于这些过程与DNA损伤综合征和癌症密切相关，我们的研究将为我们提供信息。 这些疾病的潜在机制，并帮助开发新的诊断和治疗策略。

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.