Regulation of the DNA Damage Response

描述（由申请人提供）： 项目摘要：真核细胞面临着来自内源性和环境来源的对其基因组完整性的持续挑战，并且已经进化出复杂的过程来响应和最小化长期DNA损伤。细胞利用DNA损伤耐受（DDT）途径，通过填补DNA中复制障碍或损伤产生的缺口来确保完成DNA复制。通过允许复制继续，即使面对微小的病变，DDT途径防止复制叉停止，这可能导致叉崩溃，导致严重的染色体异常和基因组不稳定。然而，至少有一个臂的DNA损伤耐受途径，称为translesion合成（TLS），可以是诱变性的，其程度取决于病变和TLS聚合酶参与。因此，从广义上考虑，DNA损伤耐受途径有助于维持基因组稳定性，但也可以促进诱变，这可能有助于癌症和耐药性。这项建议的总体目标是了解分子机制，管理损害特异性病变旁路。在上一个资助期，我们确定SHPRH和HLTF是多功能蛋白质，有助于哺乳动物细胞中DDT途径的多个方面。这些蛋白质以损伤特异性的方式发挥作用，对烷基化损伤和具有互补特异性的UV损伤作出反应，但它们的作用机制仍不明确。为了了解SHPRH和HLTF如何调节DNA损伤耐受性，我们将使用遗传和生化方法相结合来研究它们的调节及其在DDT中的作用。该提案的第一个目的将解决DNA损伤后HLTF和SHPRH如何调节。我们将分析HLTF和SHPRH与受损复制叉的相互作用，并确定不同类型的DNA损伤如何影响HLTF和SHPRH与DDT的另一个调节因子Rad18的相互作用。第二个目标是研究高级别信托基金和高级别信托基金在滴滴涕中的作用和机制。我们将询问这些蛋白质如何影响诱变和模板转换、停滞叉的蛋白质组成以及重复序列的稳定性。总的来说，这些研究将为两个关键调控基因的调控提供新的见解，这两个基因有助于最大限度地减少复制应激引起的突变。已发现HLTF和SHPRH在许多癌症中发生改变，因此这些研究与通过基因突变修饰的癌症和其他疾病高度相关。

DESCRIPTION (provided by applicant): Project Summary: Eukaryotic cells face constant challenges to the integrity of their genome from both endogenous and environmental sources, and sophisticated processes have evolved to respond to and minimize long-term DNA damage. Cells employ DNA damage tolerance (DDT) pathways to ensure the completion of DNA replication by filling in gaps created by replication barriers or lesions in the DNA. By allowing replication to continue even in the face of minor lesions, DDT pathways prevent replication forks from stalling, which can lead to fork collapse resulting in serious chromosomal abnormalities and genome instability. Nevertheless, at least one arm of the DNA damage tolerance pathway, known as translesion synthesis (TLS), can be mutagenic, the extent to which depends on the lesion and TLS polymerase involved. Thus, considered broadly, DNA damage tolerance pathways help maintain genome stability but can also promote mutagenesis, which can contribute to cancer and drug resistance. The overall goal of this proposal is to understand the molecular mechanisms that govern damage-specific lesion bypass. In the previous funding period, we identified SHPRH and HLTF as multi-functional proteins that contribute to multiple aspects of DDT pathways in mammalian cells. These proteins exert their effects in a damage-specific manner, responding to alkylation damage and UV damage with complementary specificities, yet the mechanisms by which they act remain undefined. In order to understand how SHPRH and HLTF regulate DNA damage tolerance, we will study their regulation and their role in DDT using a combination of genetic and biochemical approaches. The first aim of this proposal will address how HLTF and SHPRH are regulated following DNA damage. We will analyze the interaction of HLTF and SHPRH with damaged replication forks, and determine how different types of DNA damage affect the interaction of HLTF and SHPRH with Rad18, another regulator of DDT. The second aim will address the function and mechanism of action of HLTF and SHPRH in DDT. We will ask how these proteins affect mutagenesis and template switching, the protein composition of stalled forks, and the stability of repeat sequences. Collectively, these studies will provide novel insights into the regulation of two critical regulatory genes that help to minimize mutations arising from replication stress. Both HLTF and SHPRH have been found to be altered in numerous cancers, hence these studies are highly relevant to cancer and other diseases that are modified by genetic mutations.