Regulation of replication and recombination intermediates

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基因 DNA脆性综合征和肿瘤的潜在改变。与DNA紧密结合的非组蛋白蛋白 (蛋白质障碍)是分叉堵塞的主要原因，其中很大一部分位于重复的 核糖体DNA(RDNA)。RDNA是核仁的组织者，占不同物种基因组的10%-30%。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 具有独特的功能，例如由于高水平的rRNA转录和要求而产生更大的拓扑压力 延长复制体的维持时间。可确保rDNA复制完成的机制 挑战尚不清楚。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Smc5/6 Complex为应对rDNA的独特挑战提供了一种综合解决方案。我们发现SMC5/6是 对于完成rDNA区域的复制是必不可少的，但在非rDNA区域则不是。我们进一步确定SMC5/6 在rDNA蛋白屏障上限制复制分叉逆转。我们的新数据让我们建议Smc5/6使用 结合其亚基的活动，在rDNA蛋白屏障处调节停滞的叉子，并确保适当的rDNA 复制终止。我们计划使用分子、基因和基因的组合来验证这一中心假设 目标1中的生化方法。 当停止的复制分叉无法恢复时，崩溃的分叉和未复制的DNA缺口可以修复 通过同源重组，产生重组中间体，如Holliday连接。迅速 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，因为有丝分裂会导致后期dna纠缠。 桥、微核形成和基因组不稳定。我们和其他人的研究发现了多个 对Holliday连接移除至关重要的监管因素。然而，它们的作用机制仍然是 将被澄清。我们目前对保守的调节因子之一ESc2蛋白的研究，这是至关重要的 对于基因组的稳定性，导致了其作用机制的新模型。特别是，我们建议ESc2使用 一种促进HJ溶解的双峰策略，包括结构贡献和相扑介导的 机制。在目标2中，我们计划测试这一模型，并定义ESc2如何实现HJ清除。要完成 本提案的目标是，我们将在高效酵母系统中使用高分辨率分析。结果： 这项拟议的工作将扩展我们对几个过程的看法，包括rDNA复制完成的方式 如何以特定于上下文的方式监管复制分叉，以及重组如何起到中间作用 可以通过调节蛋白来帮助去除。因为这些过程与DNA损伤密切相关 症状和癌症，我们的研究将揭示这些疾病的潜在机制，并有助于发展 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 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.