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

DNA 损伤的修复对于防止可导致人类疾病的突变积累至关重要，例如 作为癌症。许多蛋白质对于 DNA 修复很重要，其中包括 Sgsl，一种在 DNA 中发生突变的蛋白质。 人类细胞会导致许多毁灭性疾病（即布卢姆综合症、沃纳综合症、罗斯蒙德-汤姆森综合症）， 都是癌症易感性的根本特征。 Sgsl 与一组基因相互作用 蛋白质统称为 SHU 复合体。尽管 Sgsl 已被广泛分析，但分子 其修复 DNA 损伤的机制及其与 SHU 复合物的关系仍不清楚 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。在本次K99阶段 根据建议，我将利用 Sgsl 的功能分离等位基因来描述其在 DNA 修复过程中的作用 DNA复制。我的初步结果表明，可以使用替代途径来修复 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一新途径的蛋白质，并 确定如何差异调节该途径的利用。 K99阶段的第二部分将 使用荧光显微镜将 Sgs1/Top3/Rmi1 蛋白质按照所用蛋白质组装的顺序放置 在 DNA 修复过程中，确定 Sgsl 灶形成的遗传要求是否因不同因素而异 DNA 损伤的类型。在ROO阶段，我会重点关注SHU综合体，首先分析角色 一种 SHU 成分 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 复合体通过其物理相互作用在 DNA 修复和复制过程中发挥机械作用 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 复杂并阐述了复杂形成的意义。我在K99期间接受的培训 该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。

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.