Roles of DNA Ligase 1 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。在这个提案中，我们主要关注在DNA复制和DNA修复中起关键作用的人DNA连接酶I(HLigI)。我们已经证明了hLigI与DNA滑动钳--增殖细胞核抗原和将增殖细胞核抗原装载到DNA上的载入器--RFC相互作用，并且，使用基于细胞的分析，我们已经证明了这些相互作用具有生物学意义。在第一个特定目标中，我们将阐明RFC、增殖细胞核抗原和hLigI在复制和修复过程中相互作用的功能后果，特别是hLig1和RFC之间的相互作用。HLigI还与检查点夹子及其加载器hRad17-RFC相互作用。我们的体外研究表明，hLigI和钳制加载器之间的物理和功能相互作用对hLigI的磷酸化状态很敏感。在第二个特定目标中，我们将结合定量质谱学、生化和基于细胞的分析来阐明hLigI磷酸化在调节其细胞功能中的作用。HLigI和其他人类DNA连接酶的小分子抑制剂已经通过基于结构的方法进行了鉴定。在第三个具体目标中，我们将表征hLigI小分子抑制剂的体外和体内活性。这些抑制剂不仅将作为连接反应的新探针，而且还为描述hLigI和其他人类DNA连接酶的细胞功能提供了一种有益的方法。此外，这些抑制剂的细胞抑制活性和细胞毒活性表明，它们可能作为一类新型的DNA修复抑制剂在临床上应用，可以与DNA损伤剂联合用于治疗癌症。与公共卫生相关：众所周知，基因组的不稳定推动了从正常细胞向癌细胞的进展。人类细胞有一个复杂的通路网络，这些通路共同作用来维持基因组的稳定。从机制上理解这些途径将为肿瘤抑制提供基本的见解。此外，基因组不稳定是肿瘤细胞的一个特征，这表明在正常情况下维持稳定的途径存在差异。正常细胞和癌细胞之间的这些差异为开发有选择性地针对癌细胞的治疗策略提供了机会。

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 this proposal we are focused on human DNA ligase I (hLigI) which plays a key role in DNA replication and DNA repair. We have shown that hLigI interacts with PCNA, a DNA sliding clamp, and RFC, the loader that loads PCNA onto DNA and, using cell-based assays, we have demonstrated that these interactions are biologically relevant. In the first Specific Aim, we will elucidate the functional consequences of the interactions among RFC, PCNA and hLigI during replication and repair with a particular emphasis on the interaction between hLig1 and RFC. hLigI also interacts with the checkpoint clamp and its loader, hRad17-RFC. Our in vitro studies have shown that the physical and functional interaction between hLigI and the clamp loaders are sensitive to the phosphorylation status of hLigI. In the second Specific Aim, we will elucidate the role in hLigI phosphorylation in regulating its cellular functions using a combination of quantitative mass spectrometry and biochemical and cell-based assays. Small molecule inhibitors of hLigI and the other human DNA ligases have been identified by a structure-based approach. In the third Specific Aim, we will characterize the in vitro and in vivo activities of the small molecule inhibitors of hLigI. These inhibitors will not only serve as novel probes of the ligation reaction but also provide a complimentary approach to delineating the cellular functions of hLigI and the other human DNA ligases. Furthermore, the cytostatic and cytotoxic activities of the inhibitors suggest that they may have clinical applications as a novel class of DNA repair inhibitors that can be used in combination with DNA damaging agents used to treat cancer. PUBLIC HEALTH RELEVANCE: It is well established that genomic instability drives the progression from a normal cell into a cancer cell. Human cells have a complex network of pathways that act together to maintain genome stability. A mechanistic understanding of these pathways will provide fundamental insights into tumor suppression. In addition, genomic instability is a characteristic of tumor cells, indicating that there are differences in the pathways that normally maintain stability. These differences between normal and cancer cells offer an opportunity to develop therapeutic strategies that selectively target cancer cells.