The Rtt107 interactome structures and functions

总结 完整的基因组复制需要复制机制和 许多监管因素。这些因素可以帮助复制体处理大量的模板， 障碍及其缺陷通常与癌症和DNA损伤等人类疾病有关 综合征我们的长期目标是了解复制调节蛋白如何与细胞内的蛋白质相互作用。 复制体和辅助复制。我们和其他人已经表明，进化上保守的Smc 5/6复合物是 在正常和DNA损伤条件下促进复制所必需的。在过去的两个周期中， 通过这项资助，我们在阐明这种复合物的结构方面取得了重大进展，其独特的SUMO E3 活性及其对芽殖酵母模型系统中基因组维持的多重影响。 重要的是，与目前的提议最相关的是，我们最近表明，Smc 5/6及其约束力 支架蛋白Rtt 107是一种新的辅助大复制子合成的途径。大 复制子很难复制，并且与脆性位点和其他形式的基因组突变密切相关。 不稳定研究Smc 5/6和Rtt 107促进大复制子合成的机制将有助于我们了解Smc 5/6和Rtt 107的作用机制。 拓宽我们对高风险基因组区域维持的理解。我们发现Smc 5/6和 Rtt 107与DNA聚合酶和解旋酶复合物相互作用，并有助于它们的类小泛素化。此外，委员会认为， 这些类小泛素化事件影响复制。基于这些发现，我们假设Smc 5/6和 Rtt 107直接促进复制体功能以帮助大复制子合成。通过这项工作，我们 表明Rtt 107是Smc 5/6和其他基因组维护蛋白质网络的枢纽 因素Rtt 107如何支持该网络内的交互以及每个交互如何有助于 复制是需要解决的重要问题。 在下一个融资周期中，我们建议通过研究Smc 5/6如何 和Rtt 107影响大复制子合成过程中的复制叉功能，并定义了生物化学特征 Smc 5/6和Rtt 107与DNA聚合酶和解旋酶复合物的相互作用。此外，我们将 确定DNA聚合酶和解旋酶复合物的类小泛素化如何影响起始， 复制的延伸阶段。这些研究的结果将阐明Smc 5/6、Rtt 107和 SUMO在促进大复制子合成中的完成。最后，我们将研究生物化学 Rtt 107介导的相互作用的基础和每个相互作用的功能。为了实现这些目标，我们将使用 在高效酵母系统中遗传、生物化学、生物物理方法的组合。成果 的工作将扩大我们的看法，如何大复制子合成实现，如何相扑调节 复制，以及Rtt 107相互作用组如何促进基因组复制。这些研究将 对我们理解基因组维持机制有着广泛的影响。

Summary Complete genome duplication requires a close collaboration between the replication machinery and many regulatory factors. These factors can assist replisomes in coping with large numbers of template obstacles, and their defects are frequently associated with human diseases such as cancer and DNA damage syndromes. It is our long-term goal to understand how replication regulatory proteins interact with the replisome and aid replication. We and others have shown that the evolutionarily conserved Smc5/6 complex is essential for promoting replication under normal and DNA damaging conditions. During the last two cycles of this grant, we have made significant progress in elucidating the structure of this complex, its unique SUMO E3 activity, and its multiple effects on genome maintenance in the budding yeast model system. Importantly and most relevant to the current proposal, we recently showed that Smc5/6 and its binding partner, the scaffold protein Rtt107, comprise a new pathway that aids large replicon synthesis. Large replicons are difficult to duplicate and strongly associated with fragile sites and other forms of genomic instability. Investigating the mechanisms by which Smc5/6 and Rtt107 facilitate large replicon synthesis will broaden our understanding of the maintenance of high-risk genomic regions. We found that Smc5/6 and Rtt107 interact with DNA polymerase and helicase complexes and contribute to their sumoylation. Moreover, these sumoylation events influence replication. Based on these findings, we hypothesize that Smc5/6 and Rtt107 directly promote replisome function to aid large replicon synthesis. Extending from this work, we showed that Rtt107 is the hub of a protein network composed of Smc5/6 and other genome maintenance factors. How Rtt107 supports the interactions within this network and how each interaction contributes to replication are important questions to address. In the next funding cycle, we propose to test our hypothesis stated above by examining how Smc5/6 and Rtt107 affect replication fork functions during large replicon synthesis, and define the biochemical features of the Smc5/6 and Rtt107 interactions with DNA polymerase and helicase complexes. In addition, we will determine how the sumoylation of DNA polymerase and helicase complexes affects the initiation and elongation stages of replication. Findings from these studies will shed light on the roles of Smc5/6, Rtt107, and SUMO in promoting the completion of large replicon synthesis. Finally, we will investigate the biochemical basis of Rtt107-mediated interactions and the functions of each interaction. To achieve these goals, we will use a combination of genetic, biochemical, biophysical approaches in the highly effective yeast system. Outcomes of the proposed work will expand our view on how large replicon synthesis is achieved, how SUMO regulates replication, and how the Rtt107 interactome contributes to genome duplication. As such, these studies will have broad implications in our understanding of genome maintenance mechanisms.