Studies of the Smc5/Smc6 complex in chromosomal replication

总结 基因组复制的一个重要任务是克服大量不同的障碍所造成的 通过模板损伤或DNA和蛋白质块。过去十年的研究表明， 在面对这些障碍时拯救和完成复制的机制;这些包括稳定 和重塑停滞的复制体，使复制恢复，协调各种类型的DNA 修复和利用专门的DNA聚合酶。这些过程中的缺陷导致人类衰弱 病理学，如不育，癌症和发育缺陷;因此研究这些过程， 了解这些潜在的机制，这是我们研究的目标，具有相当大的意义 对人类健康的影响。最近的研究表明，进化上保守的Smc 5/6复合物是必需的， 生长和关键的促进复制在正常和DNA损伤条件下。我们取得了 这一赠款的第一个周期在揭示这一建筑群的功能和结构方面取得了重大进展， 芽殖酵母模型系统。我们实验室和其他实验室的结果表明，Smc 5/6复合体在细胞内发挥多种作用， 在促进复制中的作用;这些包括防止毒性重组的积累 中间体，用小蛋白修饰剂SUMO修饰复制和修复蛋白，以及 与其他复制调节因子的协作。目前Smc 5/6复合体如何执行这些操作 功能还没有得到很好的理解，对这些功能的研究将揭示各种复制促进 流程.在下一个资助期内，我们建议阐明Smc 5/6复杂函数的基础， 在酵母模型系统中使用遗传、生物化学和基因组方法的组合进行复制。 在目标1中，我们将研究Smc 5/6复合体如何与另一个保守的复制系统协作 调节因子Rtt107促进复制。我们将使用DNA的全基因组定量测量 合成和复制体-DNA缔合来表征这些因子对复制的影响。我们将 还研究了Smc 5/6复合物，Rtt107和复制机制之间的物理相互作用。在aim中 2，我们将研究Smc 5/6复合物如何利用其SUMO连接酶酶活性影响Smc 5/6的表达。 复制和修复。我们已经确定了几个关键的复制和修复酶， 显示出对这种复合物的依赖性。我们将研究这些底物的sumoylation如何有助于 在不同类型的压力条件下复制。在目标3中，我们将研究靶向 Smc 5/6复合体和Rtt 107到停滞的复制叉以实现复制调节。这些拟议 这些研究对于理解复制是如何被调节以实现正常发育具有广泛的意义 所有生物的生存。

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.