Roles of Lig3 and XRCC1 Genes in Genome Stability

描述(申请人提供)：人类基因组经常受到内源性和环境DNA损害剂的攻击。如果不修复，DNA损伤将产生突变，进而可能导致癌症形成。幸运的是，DNA修复通路的复杂网络可以移除DNA损伤。为了评估暴露在环境DNA损伤剂中的生物学意义，有必要了解复杂的细胞对DNA损伤的反应细节。与保守的LIG1和LIG4基因不同，低等真核生物缺乏哺乳动物LIG3基因的同源基因，LIG3基因至少编码三种不同的多肽。有趣的是，DNA连接酶III？相关蛋白，聚(ADP-核糖)聚合酶1(PARP-1)，XRCC1和DNA聚合酶(POL)？，它们中的每一个都与碱基切除修复和DNA单链断裂的修复有关，也只在高等真核生物中发现。利用一种改进的酵母双杂交实验，我们已经鉴定了一系列在特定蛋白质-蛋白质相互作用中存在缺陷的XRCC1突变体。在特定的目标1中，我们将利用这些突变体来描述DNA连接酶III？/XRCC1与参与碱基切除和单链断裂修复的其他蛋白质之间的蛋白质-蛋白质相互作用的功能和生物学后果。最近的研究增加了DNA连接酶III？/XRCC1参与的DNA修复交易的谱系。在特定的目标2中，我们将确定DNA连接酶III？/XRCC1如何被招募到DNA核苷酸切除修复机制中，以及这是否涉及XRCC1与增殖细胞核抗原之间的相互作用。在初步研究中，我们已经确定DNA连接酶III？/XRCC1和hRad50/hMre11/NBS之间存在相互作用。在特定的目标3中，我们将确定DNA损伤如何调节这种相互作用，以及这些蛋白质是否在修复DNA双链断裂的容易出错的非同源末端连接子路径中共同作用。有趣的是，这种容易出错的途径在癌细胞中上调，可能是它们特有的基因组不稳定性的原因。在具体目标4中，我们将鉴定和表征DNA连接酶III的小分子抑制剂。我们预计这些抑制剂不仅将成为阐明LIG3基因产物的细胞功能的有价值的试剂，而且可能成为开发新型抗癌药物的先导化合物。 与公共卫生相关：众所周知，基因组的不稳定推动了从正常细胞向癌细胞的进展。人类细胞有一个复杂的通路网络，这些通路共同作用来维持基因组的稳定。从机制上理解这些途径将为肿瘤抑制提供基本的见解。此外，基因组不稳定是肿瘤细胞的一个特征，这表明在正常情况下维持稳定的途径存在差异。正常细胞和癌细胞之间的这些差异为开发有选择性地针对癌细胞的治疗策略提供了机会。

DESCRIPTION (provided by applicant): 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. 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.