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和环境来源对其基因组完整性的持续挑战，复杂的过程已经演变成对长期DNA损伤的反应并将其降至最低。细胞利用DNA损伤耐受(DDT)途径，通过填补复制障碍或DNA损伤造成的缺口来确保DNA复制的完成。通过允许复制即使面对轻微的病变也能继续，DDT通路防止复制叉子失速，这可能导致叉子崩溃，导致严重的染色体异常和基因组不稳定。然而，DNA损伤耐受途径的至少一条臂，即跨损伤合成(TLS)，可能是突变的，其程度取决于损伤和所涉及的TLS聚合酶。因此，从广义上看，DNA损伤耐受途径有助于维持基因组的稳定，但也可以促进突变，这可能导致癌症和耐药性。这项建议的总体目标是了解支配损伤特异性病变旁路的分子机制。在之前的资助期间，我们发现SHPRH和HLTF是哺乳动物细胞中参与DDT通路多方面功能的多功能蛋白质。这些蛋白质以损伤特异性的方式发挥作用，以互补的特异性响应烷基化损伤和紫外线损伤，但它们的作用机制尚不清楚。为了了解SHPRH和HLTF如何调节DNA损伤耐受性，我们将结合遗传和生化方法研究它们的调节及其在DDT中的作用。这项提案的第一个目标将解决HLTF和SHPRH在DNA损伤后如何受到调控。我们将分析HLTF和SHPRH与受损的复制叉子的相互作用，并确定不同类型的DNA损伤如何影响HLTF和SHPRH与DDT的另一个调节子Rad18的相互作用。第二个目标将阐述HLTF和SHPRH在滴滴涕中的作用和作用机制。我们将询问这些蛋白质如何影响突变和模板切换、停滞的叉子的蛋白质组成以及重复序列的稳定性。总的来说，这些研究将为调节两个关键调控基因提供新的见解，这两个基因有助于将复制应激引起的突变降至最低。HLTF和SHPRH已被发现在许多癌症中都发生了改变，因此这些研究与癌症和其他由基因突变改变的疾病高度相关。

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.