Roles of the Smc5-Smc6 Holocomplex in Genome Stability

描述（由申请人提供）： 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支持准确染色体传递的关键机制。这个建议集中在进化上保守的Smc 5-Smc 6 holocomplex和它的辅因子Rad 60;它们是基因组稳定性和DNA修复的主要调节因子。我们鉴定了Smc 5-Smc 6复合物的八个核心亚基，揭示了与相关的粘附素和凝聚素复合物不同，Smc 5-Smc 6可以通过用SUMO和/或泛素修饰其他蛋白质来调节它们的作用。此外，我们发现Rad 60通过与Ubc 9形成结构类似的非共价复合物来模拟SUMO，并且以这种方式促进Smc 5-Smc 6介导的SUMO化。这一发现解释了Smc 5-Smc 6、Rad 60和SUMO在基因组维持中观察到的功能重叠。此外，我们最近揭示了Smc 5-Smc 6在以下方面的关键但机制上未定义的功能：（i）促进MI分裂处的减数分裂染色体分离，（ii）在与Tdp 1平行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崩溃的复制叉期间产生的DNA结构的解析。在此基础上，令人信服的初步数据，我们建议阐明Smc 5-Smc 6和Rad 60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、生物化学和质谱实验来实现我们的目标。我们有两个具体目标。目标1：明确Smc 5-Smc 6在处理Holliday连接中的作用机制，Holliday连接是减数分裂和复制叉重启过程中产生的同源重组依赖性共价染色体连接。此外，还将确定Smc 5-Smc 6和Rad 60促进遗传毒性蛋白-DNA加合物（例如Top1 cc）修复的途径和蛋白质。目标二：通过以下方式定义Smc 5-Smc 6基因组稳定“网络”：（i）表征亚型Smc 5-Smc 6亚基的新型剂量抑制剂，以及（ii）测试Smc 5-Smc 6介导的类小泛素化对我们最近鉴定的靶蛋白的生理影响。虽然我们的每一个目标都是独立的，但每个目标的结果都将 可能协同提供快速洞察Smc 5-Smc 6和Rad 60的关键功能。总的来说，我们目标的完成将大大加深我们对特定基因组稳定性机制的理解，这些机制与人类疾病的病因学和治疗相关。

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.