Roles of DNA Ligase I in Mammalian DNA Metabolism

描述(申请人提供)：我们实验室的长期目标是通过研究这些不同DNA交易中常见的DNA连接步骤来阐明DNA复制、修复和重组的分子机制。人类细胞中有三个基因编码DNA连接酶。这些酶参与不同的细胞功能是由特定的蛋白质-蛋白质与不同的伙伴蛋白质相互作用所决定的。在他的建议中，我们将重点放在人类DNA连接酶I(HLigI)上，它在DNA复制和DNA修复中发挥着关键作用。值得注意的是，hLigI的缺乏和过表达都会导致基因组的不稳定。在具体目标1中，我们将重点在分子水平上定义由hLigI缺乏引起的复制叉处的异常。这些研究将提供新的见解 探讨冈崎片段的连接、核小体组装和染色质成熟之间的关系。有趣的是，hLigI活性的降低和参与维持基因组稳定性的Elg1-RFC钳制加载器的活性降低，都会导致增殖细胞核抗原与DNA的结合增加。此外，我们在初步研究中发现了hLigI与Elg1-RFC复合体之间的相互作用。在特定的目标2中，我们将检验hLigI和Elg1-RFC复合体之间的物理和功能相互作用将冈崎片段的连接与增殖细胞核抗原卸载联系起来，以及增殖细胞核抗原卸载缺陷导致核小体组装和染色质成熟异常的假设。HLigI水平升高导致基因组不稳定的观察结果为描述调节hLigI细胞水平的机制提供了令人信服的理论基础。在预赛中 研究表明，库林-DDB1泛素连接酶底物特异性因子DCAF7是一种hLigI相互作用蛋白，在DNA损伤和细胞增殖抑制的情况下，hLigI通过泛素-蛋白酶体途径降解。HLigI泛素化在调节hLigI的稳态水平以应对DNA损伤和抑制细胞增殖方面的作用将在特定的目标3中被探索。所提出的研究将为维持基因组稳定和预防癌症形成中起关键作用的途径的机制和调控提供新的见解。此外，这些信息将为表征依赖hLigI的基因组维持途径中的异常并确定它们如何促进肿瘤细胞的恶性表型提供框架。

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to elucidate the molecular mechanisms of DNA replication, repair and recombination by studying the DNA joining step that is common to these different DNA transactions. There are three genes encoding DNA ligases in human cells. The participation of these enzymes in different cellular functions is directed by specific protein-protein interactions with different partner proteins. In his proposal we are focused on human DNA ligase I (hLigI), which plays a key role in DNA replication and DNA repair. Notably, both deficiency and overexpression of hLigI cause genomic instability. In Specific Aim 1, we will focus on defining at the molecular level the abnormalities t the replication fork that are caused by hLigI deficiency. These studies will provide novel insights into the relationship between the joining of Okazaki fragments, nucleosome assembly and chromatin maturation. Interestingly, reduced hLigI activity and reduced activity of the Elg1-RFC clamp loader, which is involved in the maintenance of genomic stability, both result in increased association of PCNA with DNA. In addition, we have discovered an interaction between hLigI and the Elg1-RFC complex in preliminary studies. In Specific Aim 2, we will test the linked hypotheses that the physical and functional interplay between hLigI and the Elg1-RFC complex links the ligation of Okazaki fragments with PCNA unloading and that defects in PCNA unloading cause abnormalities in nucleosome assembly and chromatin maturation. The observation that elevated levels of hLigI cause genomic instability provides a compelling rationale for delineating the mechanisms that regulate the cellular levels of hLigI. In preliminary studies, we have identified DCAF7, a substrate specificity factor of the Cullin-DDB1 ubiquitin ligase, as a hLigI interacting protein and show that hLigI is degraded by the ubiquitin-proteasome pathway in response to both DNA damage and inhibition of cell proliferation. The role of hLigI ubiquitylation by the Cullin-DDB1 ubiquitin ligase in regulating the steady state levels of hLigI in response to both DNA damage and inhibition of cell proliferation will be explored in Specific Aim 3. The proposed studies will provide novel insights into the mechanisms and regulation of pathways that play a critical role in maintaining genome stability and preventing cancer formation. In addition, this information will provide the framework for characterizing abnormalities in hLigI-dependent genome maintenance pathways and determining how they contribute to malignant phenotype of tumor cells.