Roles of DNA Ligase 1 in Mammalian DNA Metabolism

DNA 连接酶 1 在哺乳动物 DNA 代谢中的作用

• 批准号: 8208177
• 负责人: Alan E Tomkinson
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208177
• 关键词: Address; Biochemical; Biological; Biological Assay; Cell Cycle Checkpoint; Cell Cycle Stage; Cell Proliferation; Cell Survival; Cell physiology; Cells; Characteristics; Complex; Cytostatics; DNA; DNA Damage; DNA Ligases; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA ligase I; Enzymes; Excision Repair; Genes; Genetic; Genome Stability; Genomic Instability; Goals; Health; Human; In Vitro; Laboratories; Ligation; Malignant Neoplasms; Mass Spectrum Analysis; Metabolism; Molecular; Normal Cell; Okazaki fragments; Pathway interactions; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Process; Progress Reports; Proteins; Reaction; Resolution; Role; Slide; Specificity; Structure; Testing; Therapeutic; Tumor Suppression; base; cancer cell; clinical application; cytotoxic; human DNA; in vivo; inhibitor/antagonist; insight; neoplastic cell; novel; protein protein interaction; public health relevance; recombinational repair; repaired; small molecule

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.

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 3 个编码 DNA 连接酶的基因。这些酶参与不同的细胞功能是通过与不同伙伴蛋白的特定蛋白质-蛋白质相互作用来指导的。在本提案中，我们重点关注人类 DNA 连接酶 I (hLigI)，它在 DNA 复制和 DNA 修复中发挥着关键作用。我们已经证明 hLigI 与 PCNA（DNA 滑动夹）和 RFC（将 PCNA 加载到 DNA 上的装载机）相互作用，并且使用基于细胞的测定，我们已经证明这些相互作用具有生物学相关性。在第一个具体目标中，我们将阐明复制和修复过程中 RFC、PCNA 和 hLigI 之间相互作用的功能后果，特别强调 hLig1 和 RFC 之间的相互作用。 hLigI 还与检查点夹及其加载器 hRad17-RFC 相互作用。我们的体外研究表明，hLigI 和夹钳装载机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，我们将结合定量质谱分析以及生化和细胞分析来阐明 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通过基于结构的方法鉴定出来。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。这些抑制剂不仅可以作为连接反应的新型探针，还可以提供一种互补的方法来描述 hLigI 和其他人类 DNA 连接酶的细胞功能。此外，抑制剂的细胞抑制和细胞毒性活性表明它们可能作为一类新型 DNA 修复抑制剂具有临床应用，可与用于治疗癌症的 DNA 损伤剂联合使用。公共卫生相关性：众所周知，基因组不稳定性会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的稳定性。对这些途径的机械理解将为肿瘤抑制提供基本见解。此外，基因组不稳定性是肿瘤细胞的一个特征，表明通常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。