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

描述(由申请人提供)：修复DNA损伤对于防止可能导致人类疾病(如癌症)的突变积累至关重要。许多蛋白质对DNA修复很重要，包括SGSL，这种蛋白质在人类细胞中发生突变时会导致许多破坏性疾病(即Bloom、Werner、Rothmund-Thomson综合征)，这些疾病的根本特征都是癌症易感性。SGS1在基因上与一组统称为SHU复合体的蛋白质相互作用。尽管SGSL已经被广泛分析，但它如何修复DNA损伤以及它与SHU复合体的关系的分子机制仍然不清楚，这主要是因为它的缺失导致了许多多效性表型。在这项提议的K99阶段，我将利用SGS1的一个功能分离的等位基因，它描述了它在DNA修复和DNA复制过程中的作用。我的初步结果表明，另一种途径被用来修复DNA复制错误，这与同源重组机制不同。这里提出的实验将使用遗传和细胞生物学方法来表征这一新途径中涉及的蛋白质，并确定这一途径的利用是如何受到差异调控的。K99阶段的第二部分将使用荧光显微镜将SGS1/top3/Rmi1蛋白质按DNA修复过程中使用的蛋白质组装顺序放置，并确定SGSL焦点形成的遗传要求是否因DNA损伤的类型而异。在R00阶段，我将重点研究SHU复合体，并首先分析SHU组分Shu1在rDNA修复和rDNA染色质结构中的作用。我的第二个目的是通过与srs2的物理作用来阐明shu复合体在DNA修复和复制过程中的机制作用。最后，我将确定SHU蛋白在形成复合体的情况下是否具有独特的细胞功能，并阐述复合体形成的意义。我在K99部分奖学金期间接受的培训将使我能够发展必要的技能，以便开始我自己的实验室，在那里我的最终职业目标是成为一家研究机构的终身教授。 相关性：DNA断裂的修复是细胞过程中最基本的过程之一。当DNA修复受到抑制时，细胞会积累基因突变和重排，这是癌症的标志。对DNA修复所需蛋白质的功能分析对于了解肿瘤发生的分子基础至关重要。

DESCRIPTION (provided by applicant): 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 devasting diseases (i.e. Bloom, Werner, Rothmund-Thomson syndromes), which are all fundamentally characterized by cancer predisposition. Sgs1 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 Sgs1 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 R00 phase, I will focus on the SHU complex and first analyze the role of one SHU component, Shu1, 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. Relevance: Repair of broken DNA is one of the most fundamental of cellular processes. When DNA repair is inhibited, cells can accumulate genetic mutations and rearrangements that are hallmarks of cancer. Functional analysis of proteins required for DNA repair is crucial for understanding the molecular basis of tumorigenesis.