Cellular Functions of Eukaryotic DNA Ligases

真核 DNA 连接酶的细胞功能

• 批准号: 7989620
• 负责人: Alan E Tomkinson
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7989620
• 关键词: Animal Model; Biochemical; Cell physiology; Complex; DNA; DNA Double Strand Break; DNA Ligases; DNA Repair Pathway; DNA Sequence Rearrangement; DNA biosynthesis; DNA ligase IV; DNA strand break; Defect; Double Strand Break Repair; Eukaryota; Eukaryotic Cell; Excision Repair; Funding; Genes; Genetic; Genetic Recombination; Genome; Genome Stability; Goals; Homologous Gene; Human; In Vitro; Interruption; LIG4 gene; Laboratories; Lifting; Link; Malignant Neoplasms; Mammalian Cell; Mammals; Molecular; Monitor; Mutation; Nonhomologous DNA End Joining; Organism; Pathway interactions; Play; Predisposition; Process; Proteins; Publishing; Reaction; Role; Saccharomyces cerevisiae; Site; Staging; System; Vertebral column; Yeasts; cancer cell; in vivo; insight; multicatalytic endopeptidase complex; phosphodiester; prevent; reconstitution; repaired; yeast protein

DESCRIPTION (provided by applicant): In multicellular organisms, genomic stability and integrity is maintained by the combined actions of an accurate DNA replication machinery and a complex network of DNA repair pathways. DNA joining is an essential step in DNA replication, in DNA excision repair and in the repair of DNA strand breaks. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Genetic studies have indicated that the LIG4 gene and the functionally homologous DNL4 gene of Saccharomyces cerevisiae participate in the repair of DNA double-strand breaks (DSB)s by non-homologous end joining (NHEJ). In mammalian cells, this pathway is required for genomic stability. Notably, defects in mammalian NHEJ result in the type of genetic rearrangements frequently observed in cancer cells and a predisposition to cancer. In this proposal, we will focus on delineating the molecular mechanisms of DNA ligase IV-dependent NHEJ in S. cerevisiae by a combination of in vitro and in vivo approaches. In published studies we have described physical and functional interactions among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, Pol4, FEN- 1(Rad27) and Dnl4/Lif1 complexes. Preliminary analysis of the assembly of these proteins factors at in vivo DSBs has revealed that Dnl4/Lif1 acts at an unexpectedly early stage of the NHEJ pathway. In Specific Aim 1, we will characterize the complex formed by Hdf1/Hdf2 and Dnl4/Lif1 at DNA ends and elucidate the role of Nej1 in NHEJ. In Specific Aim 2, we will characterize the complex formed by Hdf1/Hdf2, Rad50/Mre11/Xrs2 and Dnl4/Lif1 at DNA ends and determine whether the Hdf1/Hdf2 ring remains topologically linked to DNA following end joining by Hdf1/Hdf2, Rad50/Mre11/Xrs2 and Dnl4/Lif1. Finally, in Specific Aim 3, we will monitor the assembly and disassembly of NHEJ factors during the repair of an in vivo DSB and determine whether the proteasome participates in the turnover of NHEJ complexes assembled at DSB sites. Because of the conservation of NHEJ factors among eukaryotes, the results from our studies will provide insights into NHEJ in mammalian cells and contribute to an overall picture of how the repair of DSBs by NHEJ prevents the deleterious genetic changes associated with cancer cells.

描述(申请人提供)：在多细胞生物中，基因组的稳定性和完整性是由准确的DNA复制机制和复杂的DNA修复途径网络共同作用而维持的。DNA连接是DNA复制、DNA切除修复和DNA链断裂修复的重要步骤。目前已鉴定出三个编码DNA连接酶的哺乳动物基因：LIG1、LIG3和LIG4。遗传学研究表明，酿酒酵母的LIG4基因和功能同源的DNL4基因通过非同源末端连接参与了S双链断裂的修复。在哺乳动物细胞中，这一途径是基因组稳定所必需的。值得注意的是，哺乳动物NHEJ的缺陷导致了在癌细胞中经常观察到的基因重排类型和癌症的易感性。在这项研究中，我们将通过体外和体内相结合的方法来描述酿酒酵母中依赖DNA连接酶IV的NHEJ的分子机制。在已发表的研究中，我们描述了Hdf1/Hdf2、Rad50/Mre11/Xrs2、Pol4、Fen-1(Rad27)和Dn14/Lif1复合体之间的物理和功能相互作用。对这些蛋白质因子在体内DSB组装的初步分析表明，Dn14/Lif1在NHEJ途径的意外早期发挥作用。在特定的目标1中，我们将表征Hdf1/Hdf2和Dn14/Lif1在DNA末端形成的复合体，并阐明Nej1在NHEJ中的作用。在特定目标2中，我们将表征由Hdf1/Hdf2、Rad50/Mre11/Xrs2和Dnl4/Lif1在DNA末端形成的复合体，并确定Hdf1/Hdf2、Rad50/Mre11/Xrs2和Dnl4/Lif1末端连接后，Hdf1/Hdf2环是否保持与DNA的拓扑连接。最后，在特定的目标3中，我们将监测在体内DSB修复过程中NHEJ因子的组装和拆解，并确定蛋白酶体是否参与在DSB位点组装的NHEJ复合体的周转。由于NHEJ因子在真核生物中的保守，我们的研究结果将为哺乳动物细胞中的NHEJ提供见解，并有助于全面了解NHEJ如何修复DSB防止与癌细胞相关的有害基因变化。