Cellular Functions of Eukaryotic DNA Ligases

描述（由申请人提供）：在多细胞生物中，基因组的稳定性和完整性是通过精确的DNA复制机制和复杂的DNA修复途径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链断裂修复的重要步骤。编码DNA连接酶的三个哺乳动物基因LIG1、LIG3和LIG4已经被鉴定出来。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（non-homologous end joining， NHEJ）参与DNA双链断裂（DSB）的修复。在哺乳动物细胞中，这种途径是基因组稳定所必需的。值得注意的是，哺乳动物NHEJ的缺陷导致癌症细胞中常见的基因重排和癌症易感性。在本研究中，我们将通过体外和体内相结合的方法，重点描述酿酒葡萄球菌DNA连接酶iv依赖性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Dnl4/Lif1复合物之间的物理和功能相互作用。对这些蛋白因子在体内dsb中组装的初步分析表明，dn14 /Lif1在NHEJ通路的早期出乎意料地起作用。在Specific Aim 1中，我们将描述Hdf1/Hdf2和Dnl4/Lif1在DNA末端形成的复合物，并阐明Nej1在NHEJ中的作用。在Specific Aim 2中，我们将表征Hdf1/Hdf2、Rad50/Mre11/Xrs2和Dnl4/Lif1在DNA末端形成的复合物，并确定Hdf1/Hdf2环在Hdf1/Hdf2、Rad50/Mre11/Xrs2和Dnl4/Lif1末端连接后是否仍与DNA保持拓扑连接。最后，在Specific Aim 3中，我们将监测体内DSB修复过程中NHEJ因子的组装和拆卸，并确定蛋白酶体是否参与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，我们的研究结果将提供对哺乳动物细胞中NHEJ的见解，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.