Mechanistic insights into the SHU complex and Sgs1 in DNA repair and replication

DNA损伤的修复对于防止可能导致人类疾病的突变的积累至关重要，例如 就像癌症一样。许多蛋白质对DNA修复很重要，包括SGSL，一种当突变时 人类细胞导致许多吞噬疾病(即布鲁姆、沃纳、罗斯蒙德-汤姆森综合征)，这些疾病 都是以癌症易感性为基本特征的。SGSL与一组 蛋白质统称为SHU复合体。尽管SGSL已经被广泛分析，但分子 它修复DNA损伤的机制及其与SHU复合体的关系仍然存在 难以捉摸，很大程度上是因为它的缺失导致了许多多效性表型。在此的K99阶段 提议，我将利用SGSL的一个功能分离的等位基因，它描述了它在DNA修复中的作用 DNA复制。我的初步结果表明，另一种途径被用来修复DNA复制 与同源重组机制不同的误差。这里提出的实验将 使用遗传和细胞生物学方法来表征参与这一新途径的蛋白质和 确定这一途径的利用是如何进行差异化调控的。K99阶段的第二部分将 使用荧光显微镜将SGS1/top3/Rmi1蛋白按所用蛋白组装的顺序排列 在DNA修复过程中，并确定SGSL焦点形成的遗传要求是否根据不同的 DNA损伤的类型。在Roo阶段，我将重点介绍SHU Complex，并首先分析其角色 SHU组分Shul在rDNA修复和rDNA染色质结构中的作用。我的第二个目标将阐明 SHU复合体通过物理作用在DNA修复和复制中的机制作用 与srs2。最后，我将确定SHU蛋白是否具有独特的细胞功能，尽管形成了 并阐述了复合体形成的重要性。我在K99期间接受的训练 奖学金的一部分将使我能够发展必要的技能，开始我自己的实验室，在那里我的 最终的职业目标是成为一家研究机构的终身教授。

Repair of DNA damage is crucial to prevent accumulation of mutations that can cause human disease, such as cancer. Many proteins are important for DNA repair including Sgsl, a protein that when mutated in human cells leads to many devesting diseases (i.e. Bloom, Werner, Rothmund-Thomson syndromes), which are all fundamentally characterized by cancer predisposition. Sgsl genetically interacts with a group of proteins collectively called the SHU complex. Although Sgsl has been extensively analyzed, the molecular mechanism of how it functions to repair DNA damage and its relationship to the SHU complex has remained elusive, largely because its deletion leads to many pleiotropic phenotypes. During the K99 phase of this proposal, I will utilize a separation-of-function allele of Sgsl that delineates its role during DNA repair from DNA replication. My preliminary results suggest that an alternative pathway is used to repair DNA replication errors that is distinct from the homologous recombination machinery. The experiments proposed here will use genetic and cell biological approaches to characterize the proteins involved in this novel pathway and determine how utilization of this pathway is differentially regulated. The second part of the K99 phase will use flourescent microscopy to place the Sgs1/Top3/Rmi1 proteins in the order of protein assembly utilized during DNA repair and determine if the genetic requirements for Sgsl foci formation differ depending upon the type of DNA damage. During the ROO phase, I will focus on the SHU complex and first analyze the role of one SHU component, Shul, in rDNA repair and rDNA chromatin structure. My second aim will elucidate the mechanistic role of the SHU complex during DNA repair and replication through its physical interaction with Srs2. Finally, I will determine if the SHU proteins have unique cellular functions despite forming a complex and address the significance of complex formation. The training that I receive during the K99 portion of the fellowship will enable me to develop the skills necessary to begin my own laboratory where my ultimate career goal is to be a tenured professor at a research institution.