Roles of DNA Ligase 1 in Mammalian DNA Metabolism

描述（由申请人提供）：DNA链断裂是DNA复制、DNA修复和基因重组途径中的中间产物。连接磷酸二酯主干中断的能力对基因组的完整性和稳定性至关重要。已经确定了三个编码DNA连接酶的人类基因。有令人信服的遗传和生物化学证据表明，人类LIG1基因的产物，DNA连接酶I，与DNA复制和DNA切除修复有关。此外，在小鼠模型中，在一个症状包括发育异常、免疫缺陷和淋巴瘤的人类个体的LIG1基因中发现了一个突变，这证明了DNA连接酶I缺乏会导致遗传不稳定和易患癌症。以前，我们已经描述了人类DNA连接酶I和复制滑动钳PCNA之间的物理和功能相互作用。由于这种相互作用不会刺激DNA连接，我们寻找可能在连接步骤中起作用的其他因素。在初步研究中，我们已经检测到与复制夹夹加载器RFC的物理和功能相互作用，并表明在功能同源的酵母蛋白、Cdc9 DNA连接酶、yRFC和yPCNA （Pol30）之间也存在类似的相互作用。在Specific Aim 1中，我们将阐明这些成对相互作用协调冈崎片段连接的分子机制。在特异性目标2中，我们将研究磷酸化在调节DNA连接酶I和RFC之间的功能相互作用中的作用。我们还发现DNA连接酶I与含有hRad17和hRad9-hRad1-hHus1复合物的夹钳装载复合物相互作用，这表明DNA连接酶I与DNA损伤和复制阻断激活的细胞周期检查点之间存在一种新的意想不到的联系。在Specific Aim 3中，我们将描述DNA连接酶I和检查点夹加载器/夹复合物之间的相互作用。这些研究的长期目标是了解DNA连接酶I在DNA复制和修复中的分子机制。

DESCRIPTION (provided by applicant): DNA strand breaks occur as intermediates in pathways of DNA replication, DNA repair and genetic recombination. The ability to join interruptions in the phosphodiester backbone is critical for genome integrity and stability. Three human genes encoding DNA ligases have been identified. There is compelling genetic and biochemical evidence linking the product of the human LIG1 gene, DNA ligase I, with DNA replication and DNA excision repair. Moreover, the reiteration of a mutation identified in the LIG1 gene of a human individual, whose symptoms included developmental abnormalities, immunodeficiency and lymphoma, in a mouse model has proven that DNA ligase I deficiency causes genetic instability and predisposition to cancer. Previously we have characterized a physical and functional interaction between human DNA ligase I and the replicative sliding clamp, PCNA. Since this interaction did not stimulate DNA joining, we searched for additional factors that may act at the ligation step. In preliminary studies we have detected a physical and functional and functional interaction with the replicative clamp loader RFC and shown that similar interactions occur among the functionally homologous yeast proteins, Cdc9 DNA ligase, yRFC and yPCNA (Pol30). In Specific Aim 1, we will elucidate the moelcular mechanisms by which these pairwise interactions co-ordinate Okazaki fragment joining. In Specific Aim 2, we will examine of phosphorylation in regulating the functional interaction between DNA ligase I and RFC. We have also found that DNA ligase I interacts with an alternative clamp loader complex containing hRad17 and an alternative clamp, hRad9-hRad1-hHus1 complex, suggesting a novel and unexpected link between DNA ligase I and cell cycle checkpoints activated by DNA damage and replication block. In Specific Aim 3, we will characterize the interactions between DNA ligase I and the checkpoint clamp loader/clamp complexes. The long term goal of these studies is to understand the molecular mechanisms by which DNA ligase I functions in DNA replication and repair.