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

DNA 修复和复制中 SHU 复合物和 Sgs1 的机制见解

基本信息

批准号：
8655759
负责人：
Kara A Bernstein
金额：
$ 0.35万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2014-06-30
项目状态：
已结题

项目摘要

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.

修复DNA损伤对于防止可能导致人类疾病的突变积累至关重要，癌症许多蛋白质对于DNA修复是重要的，包括Sgsl，一种当在细胞中突变时，人类细胞导致许多发育不良性疾病（即Bloom，Werner，Rothmund-Thomson综合征），都有患癌倾向的基本特征Sgsl在遗传上与一组这些蛋白质统称为SHU复合物。尽管Sgsl已经被广泛分析，但其分子量它如何修复DNA损伤的机制及其与SHU复合物的关系仍然存在这是一个难以捉摸的基因，很大程度上是因为它的缺失导致许多多效性表型。在K99阶段，根据这项提议，我将利用Sgsl的一个功能分离等位基因，描述其在DNA修复过程中的作用， DNA复制。我的初步结果表明，一个替代途径是用来修复DNA复制与同源重组机制不同的错误。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种新途径的蛋白质，确定如何利用这一途径的差异调节。K99阶段的第二部分将使用荧光显微镜将Sgs 1/Top3/Rmi 1蛋白按照所用的蛋白组装顺序放置并确定Sgsl灶形成的遗传要求是否取决于 DNA损伤的类型在ROO阶段，我将重点关注SHU情结，首先分析其作用 SHU的一个组成部分，Shul，在rDNA修复和rDNA染色质结构。我的第二个目标将阐明 SHU复合物通过其物理相互作用在DNA修复和复制过程中的机械作用关于SRS 2最后，我将确定SHU蛋白质是否具有独特的细胞功能，尽管它们形成了一个细胞膜。复杂性和复杂性形成的意义。我在K99期间接受的培训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，最终的职业目标是成为研究机构的终身教授。