Roles of DNA Ligase I in Mammalian DNA Metabolism

描述（由申请人提供）：我们实验室的长期目标是通过研究这些不同DNA交易中共同的DNA连接步骤来阐明DNA复制、修复和重组的分子机制。人类细胞中有三种基因编码DNA连接酶。这些酶参与不同的细胞功能是由特定的蛋白质与不同的伴侣蛋白相互作用指导的。在他的建议中，我们专注于人类DNA连接酶I (hLigI)，它在DNA复制和DNA修复中起着关键作用。值得注意的是，hLigI缺乏和过表达都会导致基因组不稳定。在Specific Aim 1中，我们将重点在分子水平上定义由hLigI缺陷引起的复制叉的异常。这些研究将提供新的见解

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.