Roles of Lig3 and XRCC1 Genes in Genome Stability

摘要 人类基因组不断受到内源性和环境DNA的攻击 破坏剂。如果不修复，DNA损伤将引起突变，反过来可能导致 癌症形成幸运的是，一个复杂的DNA修复途径网络可以清除 DNA损伤评估暴露于环境DNA损伤的生物学意义 因此，有必要了解细胞对DNA的复杂反应的细节。 损害与保守的LIG 1和LIG 4基因不同，低等真核生物缺乏LIG 1和LIG 4基因的同源物。 哺乳动物LIG 3基因，其编码至少三种不同的多肽。有趣的是 DNA连接酶III相关蛋白，聚（ADP-核糖）聚合酶1（PARP-1），XRCC 1和 DNA聚合酶（Pol）<$，其中每一种都涉及碱基切除修复和DNA聚合酶。 DNA单链断裂的修复也仅在高等真核生物中发现。使用修改的 酵母双杂交试验，我们已经确定了一系列的XRCC 1突变体是有缺陷的， 特异性蛋白质间相互作用。在具体目标1中，我们将利用这些突变体来描述 DNA连接酶之间蛋白质-蛋白质相互作用的功能和生物学后果 III <$/XRCC 1和其他参与碱基切除和单链断裂修复的蛋白质。最近 研究已经增加了DNA修复处理的全部功能，其中DNA连接酶 III <$/XRCC 1参与。在特定目标2中，我们将确定DNA连接酶III <$/XRCC 1是如何 招募到DNA核苷酸切除修复机制，以及这是否涉及 XRCC 1与PCNA相互作用。在初步研究中，我们已经确定了一种相互作用 DNA连接酶III <$/XRCC 1和hRad 50/hMre 11/Nbs之间的差异。在具体目标3中，我们将确定 DNA损伤是如何调节这种相互作用的，以及这些蛋白质是否错误地共同作用- 修复DNA双链断裂的倾向性非同源末端连接子途径。 有趣的是，这种易出错的途径在癌细胞中上调，并可能有助于它们的生长。 基因组的不稳定性在具体目标4中，我们将识别和描述小 DNA连接酶III的分子抑制剂。我们设想，这些抑制剂不仅会 用于阐明LIG 3基因产物的细胞功能的有价值的试剂， 作为开发新型抗癌剂的先导化合物。

Abstract The human genome is subject to constant attack by endogenous and environmental DNA damaging agents. If unrepaired, DNA lesions will give rise to mutations that in turn may lead to cancer formation. Fortunately, a complex network of DNA repair pathways operates to remove DNA lesions. To assess the biological significance of exposure to environmental DNA damaging agents, it is necessary to understand the details of the complex cellular response to DNA damage. Unlike the conserved LIG1 and LIG4 genes, lower eukaryotes lack a homolog of the mammalian LIG3 gene, which encodes at least three distinct polypeptides. Interestingly, the DNA ligase III¿-associated proteins, poly (ADP-ribose) polymerase 1 (PARP-1), XRCC1 and DNA polymerase (Pol) ¿, each of which have been implicated in base excision repair and the repair of DNA single strand breaks, are also found only in higher eukaryotes. Using a modified yeast two hybrid assay, we have identified a series of XRCC1 mutants that are defective in specific protein-protein interactions. In Specific Aim 1, we will utilize these mutants to delineate the functional and biological consequences of protein-protein interactions between DNA ligase III¿/XRCC1 and other proteins involved in base excision and single strand break repair. Recent studies have increased the repertoire of DNA repair transactions in which DNA ligase III¿/XRCC1 participates. In Specific Aim 2, we will determine how DNA ligase III¿/XRCC1 is recruited to the DNA nucleotide excision repair machinery and whether this involves an interaction between XRCC1 and PCNA. In preliminary studies, we have identified an interaction between DNA ligase III¿/XRCC1 and hRad50/hMre11/Nbs. In Specific Aim 3, we will determine how DNA damage regulates this interaction and whether these proteins act together in an error- prone non-homologous end-joining sub pathway that repairs DNA double strand breaks. Interestingly, this error-prone pathway is up-regulated in cancer cells and may contribute to their characteristic genomic instability. In Specific Aim 4, we will identify and characterize small molecule inhibitors of DNA ligase III. We envision that that these inhibitors will not only be valuable reagents for elucidating the cellular functions of the LIG3 gene products but also may serve as lead compounds for the development of novel anti-cancer agents.