Studies of the Smc5/Smc6 complex in chromosomal replication

描述(由申请人提供)： 基因组复制的一个重要任务是克服模板损伤或DNA和蛋白质块造成的大量不同障碍。过去十年的研究表明，面对这些障碍，存在多种拯救和完成复制的机制；这些机制包括稳定和重塑停滞的复制体以允许复制恢复，协调各种类型的dna修复，以及利用专门的 DNA聚合酶。这些过程中的缺陷会导致人类衰弱的病理，如不孕、癌症和发育缺陷；因此，研究这些过程并了解其潜在的机制，这是我们研究的目标，对人类健康具有相当重要的意义。最近的研究表明，进化上保守的Smc5/6复合体对于生长是必不可少的，对于在正常和DNA损伤条件下促进复制至关重要。在这项资助的第一个周期中，我们在揭示萌芽酵母模型系统中这种复合体的功能和结构方面取得了重大进展。我们的实验室和其他实验室的结果表明，Smc5/6复合体在促进复制方面发挥着多种作用，包括防止有毒重组中间体的积累，用小蛋白修饰剂SUMO修饰复制和修复蛋白，以及与其他复制调控因子合作。目前，Smc5/6复合体如何执行这些功能还不是很清楚，对这些功能的研究将有助于揭示各种促进复制的过程。在下一个授权期，我们建议在酵母模型系统中结合遗传、生化和基因组方法来阐明Smc5/6复合体在复制中的功能基础。在目标1中，我们将研究Smc5/6复合体如何与另一个保守的复制调控因子Rtt107合作促进复制。我们将使用DNA合成和复制体-DNA关联的全基因组定量测量来表征这些因素对复制的影响。我们还将研究Smc5/6复合体、Rtt107和复制机制之间的物理相互作用。在目标2中，我们将研究Smc5/6复合体如何利用其相扑连接酶活性来影响复制和修复。我们已经确定了几个关键的复制和修复酶，它们的总甲基化显示出对这个复合体的依赖。我们将研究这些底物的总和如何在不同类型的胁迫条件下促进复制。在目标3中，我们将研究针对Smc5/6复合体和Rtt107到停滞复制分叉的机制，以实现复制调控。这些拟议的研究对于理解如何调控复制以使所有生物体能够正常发育和生存具有广泛的意义。

DESCRIPTION (provided by applicant): Summary An important task of genome duplication is to overcome the large number of diverse obstacles posed by template lesions or DNA and protein blocks. Studies in the past decade suggest the existence of multiple mechanisms to rescue and complete replication in the face of these impediments; these include stabilization and remodeling of stalled replisomes to allow replication resumption, coordination of various types of DNA repair, and utilization of specialized DNA polymerases. Defects in these processes lead to debilitating human pathologies such as sterility, cancer, and developmental defects; thus studying these processes and understanding the underlying mechanisms, which are the goals of our studies, are of considerable significance to human health. Recent studies have shown that the evolutionarily conserved Smc5/6 complex is essential for growth and critical for promoting replication under normal and DNA damaging conditions. We have made significant progress in the first cycle of this grant in revealing the function and structure of this complex in the budding yeast model system. Results from our lab and others suggest that the Smc5/6 complex plays multiple roles in promoting replication; these include the prevention of accumulation of toxic recombination intermediates, the modification of replication and repair proteins with the small protein modifier SUMO, and collaboration with other replication regulatory factors. Currently how the Smc5/6 complex carries out these functions is not well understood, and research into these will shed light on the various replication-promoting processes. In the next grant period, we propose to elucidate the basis of the Smc5/6 complex function in replication using a combination of genetic, biochemical, and genomic approaches in the yeast model system. In Aim 1, we will investigate how the Smc5/6 complex collaborates with another conserved replication regulatory factor Rtt107 to promote replication. We will use quantitative genome-wide measurements of DNA synthesis and replisome-DNA association to characterize the effects of these factors on replication. We will also examine the physical interactions between the Smc5/6 complex, Rtt107, and replication machinery. In Aim 2, we will investigate how the Smc5/6 complex utilizes its SUMO ligase enzymatic activity to influence replication and repair. We have identified several key replication and repair enzymes whose sumoylation shows dependence on this complex. We will examine how the sumoylation of these substrates contributes to replication under different types of stress conditions. In Aim 3, we will investigate the mechanisms that target the Smc5/6 complex and Rtt107 to stalled replication forks to enable replication regulation. These proposed studies have broad implications for understanding how replication is regulated to enable proper development and survival of all organisms.