Cellular Functions of Eukaryotic DNA Ligases

描述（由申请人提供）：在多细胞生物体中，基因组的稳定性和完整性是通过精确的DNA复制机制和DNA修复途径的复杂网络的联合作用来维持的。DNA连接是DNA复制、DNA切除修复和DNA链断裂修复中的重要步骤。已经鉴定了三种编码DNA连接酶的哺乳动物基因，LIG 1、LIG 3和LIG 4。遗传学研究表明，LIG 4基因和酿酒酵母的功能同源的DNL 4基因通过非同源末端连接（NHEJ）参与DNA双链断裂（DSB）的修复。在哺乳动物细胞中，该途径是基因组稳定性所必需的。值得注意的是，哺乳动物NHEJ中的缺陷导致在癌细胞中经常观察到的遗传重排类型和癌症易感性。在本研究中，我们将着重于阐明S.通过体外和体内方法的组合，在酿酒酵母中表达。在已发表的研究中，我们描述了Hdf 1/Hdf 2，Rad 50/Mre 11/Xrs 2，Pol 4，FEN- 1（Rad 27）和Dnl 4/Lif 1复合物之间的物理和功能相互作用。这些蛋白质因子在体内DSB的组装的初步分析揭示了Dnl 4/Lifl在NHEJ途径的出乎意料的早期阶段起作用。在特异性目的1中，我们将描述Hdf 1/Hdf 2和Dnl 4/Lif 1在DNA末端形成的复合物，并阐明Nej 1在NHEJ中的作用。在具体目标2中，我们将表征由Hdf 1/Hdf 2、Rad 50/Mre 11/Xrs 2和Dnl 4/Lif 1在DNA末端形成的复合物，并确定Hdf 1/Hdf 2、Rad 50/Mre 11/Xrs 2和Dnl 4/Lif 1末端连接后Hdf 1/Hdf 2环是否仍与DNA拓扑连接。最后，在具体目标3中，我们将监测在体内DSB修复过程中NHEJ因子的组装和分解，并确定蛋白酶体是否参与在DSB位点组装的NHEJ复合物的周转。由于NHEJ因子在真核生物中的保守性，我们的研究结果将为哺乳动物细胞中的NHEJ提供深入了解，并有助于全面了解NHEJ如何修复DSB以防止与癌细胞相关的有害遗传变化。

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.