Roles of DNA Ligase 1 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DNA连接步骤来阐明DNA复制、修复和重组的分子机制，该步骤是这些不同DNA交易的共同点。在人类细胞中有三种编码DNA连接酶的基因。这些酶参与不同的细胞功能是由特定的蛋白质-蛋白质相互作用与不同的伴侣蛋白。在这个建议中，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。我们已经表明，hLigI与PCNA，DNA滑动钳，RFC，装载PCNA到DNA上的加载器相互作用，并使用基于细胞的测定，我们已经证明，这些相互作用是生物相关的。在第一个具体目标，我们将阐明RFC，PCNA和hLigI之间的相互作用在复制和修复过程中的功能后果，特别强调hLig 1和RFC之间的相互作用。hLigI还与检查点钳及其加载器hRad 17-RFC相互作用。我们的体外研究表明，hLigI和钳加载器之间的物理和功能相互作用对hLigI的磷酸化状态敏感。在第二个具体目标中，我们将阐明hLigI磷酸化在调节其细胞功能中的作用，使用定量质谱和生物化学和细胞为基础的测定相结合。hLigI和其他人类DNA连接酶的小分子抑制剂已通过基于结构的方法鉴定。在第三个具体目标中，我们将表征hLigI的小分子抑制剂的体外和体内活性。这些抑制剂将不仅作为连接反应的新探针，而且还提供了一种互补的方法来描绘hLigI和其他人类DNA连接酶的细胞功能。此外，抑制剂的细胞生长抑制和细胞毒性活性表明，它们可能具有作为一类新的DNA修复抑制剂的临床应用，所述DNA修复抑制剂可以与用于治疗癌症的DNA损伤剂组合使用。公共卫生相关性：众所周知，基因组不稳定性驱动正常细胞发展为癌细胞。人类细胞具有复杂的通路网络，这些通路共同作用以维持基因组稳定性。对这些途径的机制性理解将为肿瘤抑制提供基本见解。此外，基因组的不稳定性是肿瘤细胞的一个特征，这表明正常情况下维持稳定性的途径存在差异。正常细胞和癌细胞之间的这些差异为开发选择性靶向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.