Roles of the Smc5-Smc6 Holocomplex in Genome Stability

描述(由申请人提供)： 项目摘要/摘要染色体的忠实繁殖对于抑制非整倍体相关的出生缺陷和癌症至关重要。因此，我们的首要目标是确定DNA修复、复制和细胞周期途径中的关键机制，以支持准确的染色体传递。这一建议集中在进化上保守的Smc5-Smc6完整复合体及其辅因子Rad60；它们是基因组稳定性和DNA修复的主要调节因子。我们确定了Smc5-Smc6复合体的8个核心亚基，揭示了Smc5-Smc6与相关的粘附素和凝集素复合体不同，Smc5-Smc6可以通过用相扑和/或泛素修饰其他蛋白质来调节它们的作用。此外，我们还发现Rad60通过与Ubc9形成结构相似的非共价复合体来模拟相扑，从而促进了Smc5-Smc6介导的SUMO化。这一发现解释了观察到的Smc5-Smc6、Rad60和相扑在基因组维护方面的功能重叠。此外，我们最近揭示了Smc5-Smc6在(I)促进MI分裂处的减数分裂染色体分离，(Ii)在平行于Tdp1的途径中处理蛋白质-DNA加合物，以及(Iii)通过基于同源重组的修复重新启动折叠的复制叉时产生的DNA结构的关键但机制未确定的功能。在此基础上，结合令人信服的初步数据，我们建议阐明Smc5-Smc6和Rad60在这些特定的染色体分离和DNA修复过程中的机制。我们将通过整合遗传学、生物化学和质谱学实验来实现我们的目标，在经过验证的裂变酵母模式生物中进行。我们有两个具体目标。目的1：明确Smc5-Smc6处理Holliday连接的机制(S)，Holliday连接是减数分裂和复制分叉重新启动过程中产生的同源重组依赖的共价染色体连接。此外，还将确定Smc5-Smc6和Rad60促进遗传毒性蛋白-DNA加合物(例如Top1cc)修复的途径和蛋白质。目的：通过(I)鉴定新的亚型Smc5-Smc6亚基的剂量抑制因子，以及(Ii)检测Smc5-Smc6介导的Smc5-Smc6对靶蛋白的生理影响，以确定Smc5-Smc6基因组稳定的“网络”。尽管我们的每个目标都是独立的，但每个目标的结果都是 可能会协同作用，提供对Smc5-Smc6和Rad60关键功能的快速洞察。总体而言，完成我们的目标将显著加深我们对特定基因组稳定机制的理解，这些机制与人类疾病的病因和治疗都有关。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT The faithful propagation of chromosomes is crucial to suppress aneuploidy-related birth defects, and cancer. Therefore, our overarching objective is to define key mechanisms within the DNA repair, replication and cell cycle pathways that support accurate chromosome transmission. This proposal centers on the evolutionarily conserved Smc5-Smc6 holocomplex and its cofactor, Rad60; which are master regulators of genome stability and DNA repair. We identified the eight core subunits of the Smc5-Smc6 complex, revealing that unlike the related cohesin and condensin complexes, Smc5-Smc6 can regulate the action of other proteins by modifying them with SUMO and/or ubiquitin. In addition, we discovered that Rad60 mimics SUMO by forming a structurally analogous non-covalent complex with Ubc9, and in this way facilitates Smc5-Smc6-mediated SUMOylation. This discovery explains the observed functional overlap between Smc5-Smc6, Rad60 and SUMO in genome maintenance. Furthermore, we have recently revealed critical but mechanistically undefined functions for Smc5-Smc6 in (i) promoting meiotic chromosome segregation at the MI division, (ii) the processing of protein-DNA adducts in a pathway parallel to Tdp1, and (iii) the resolution of DNA structures arising during the restart of collapsed replication forks through homologous recombination-based repair. Building on this foundation and compelling preliminary data, we propose to elucidate the mechanisms of Smc5- Smc6 and Rad60 in these specific chromosome segregation and DNA repair processes. We will achieve our goal by integrating genetics, biochemistry and mass spectrometry experiments in the proven fission yeast model organism. We have two Specific Aims. Aim 1: To define the mechanism(s) of Smc5-Smc6 in processing Holliday junctions, which are homologous recombination-dependent covalent chromosome linkages generated during meiosis and replication fork restart. Also, the pathways and proteins through which Smc5-Smc6 and Rad60 promote the repair of genotoxic protein-DNA adducts (e.g. Top1cc) will be determined. Aim 2: To define the Smc5-Smc6 genome stabilizing "network" through: (i) characterization of novel dosage suppressors of hypomorphic Smc5-Smc6 subunits, and (ii) testing the physiological impact of Smc5-Smc6-mediated sumoylation on target proteins that we recently identified. Although each of our Aims is self-standing, the results from each will likely synergize to provide rapid insight into the critical functions of Smc5-Smc6 and Rad60. Overall, completion of our Aims will significantly deepen our understanding of specific genome stability mechanisms, which are relevant to both the etiology and treatment of human disease.