Studies of the Smc5/Smc6 complex in chromosomal replication

摘要 基因组复制的一项重要任务是克服大量不同的障碍 通过模板损伤或DNA和蛋白质块。过去十年的研究表明，存在多个 在这些障碍面前挽救和完成复制的机制；这些障碍包括稳定 和重塑停滞的复制体，以允许复制恢复，协调各种类型的DNA 专门化DNA聚合酶的修复和利用。这些过程中的缺陷会导致人类虚弱 病理学，如不孕不育、癌症和发育缺陷；因此研究这些过程和 理解潜在的机制，这是我们研究的目标，具有相当重要的意义。 为人类健康干杯。最近的研究表明，进化上保守的Smc5/6复合体对于 在正常和DNA损伤的条件下，生长对促进复制至关重要。我们已经做出了 在这笔赠款的第一个周期中，在揭示这一复合体在 芽殖酵母模型系统。我们的实验室和其他实验室的结果表明，Smc5/6复合体发挥着多重作用 在促进复制方面的作用；其中包括防止有毒重组的积累 中间体，复制和修复蛋白的小蛋白修饰剂相扑的修饰，以及 与其他复制监管因素的协作。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机制 流程。在下一个授权期，我们建议阐明Smc5/6复合体功能的基础 在酵母模型系统中使用遗传、生化和基因组方法的组合进行复制。 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协同工作 调节因子Rtt107促进复制。我们将使用全基因组的DNA定量测量 合成和复制体-DNA结合，以表征这些因素对复制的影响。我们会 还要研究Smc5/6复合体、Rtt107和复制机制之间的物理相互作用。在AIM 2，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。我们已经确定了几种关键的复制和修复酶，它们的总甲基化 显示出对这个情结的依赖。我们将研究这些底物的和甲基化如何有助于 在不同类型的胁迫条件下复制。在目标3中，我们将研究针对 Smc5/6复合体和Rtt107到停滞的复制分叉，以实现复制调节。这些建议 研究对于理解复制是如何被调控以实现正常发育具有广泛的意义 以及所有有机体的生存。

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.