ANALYSIS OF A DNA DAMAGE INDUCIBLE CHECKPOINT COMPLEX

细胞持续经历由复制错误、代谢诱导的氧化损伤和外源性诱变剂引起的DNA损伤。 DNA损伤触发协调的细胞反应，包括修复机制的动员和细胞周期检查点的激活，这将细胞阻滞在G1和G2/M期，并减缓S期的进展。 遗传和药理学数据表明，在酵母和人类等多种生物体中，检查点功能的破坏导致遗传不稳定性，这与哺乳动物中致癌突变的获得相关。 酵母的遗传学研究表明，检查点基因的失活可以防止DNA损伤诱导的细胞周期停滞，并使酵母对遗传毒素敏感。 虽然许多酵母检查点基因已被确定，我们的理解DNA损伤诱导的检查点激活哺乳动物已经落后。 为了鉴定哺乳动物DNA损伤反应的潜在调节因子，我们克隆了hRad 1、hRad 9和hHus 1，它们是S. pombe检查点基因，rad 1，rad 9和hus 1，我们开始了一项研究，以检查这些蛋白质在哺乳动物中的生化和细胞功能。 我们的研究结果表明，这些蛋白质在人类细胞中形成复合物。复合物的两个成员hRad 9和hRad 1被磷酸化并与染色质结合以响应DNA损伤，从而证明这些蛋白质形成损伤响应性检查点复合物。 此外，我们提供了新的数据表明，DNA损伤引起的hRad 9重新分配到核灶。 我们现在建议扩展这些发现，并定义hRad 9的生化功能，并确定其在细胞检查点反应中的作用。 该项目的具体目的是：1）确定DNA损伤后hRad 9的核保留是否反映了受损DNA的生化传感机制; 2）分析hRad 9在DNA损伤后检查点信号激活和细胞周期阻滞中的作用; 3）鉴定基础和DNA损伤诱导的hRad 9磷酸化位点，并确定它们在介导与hRad 1/hRad 9相互作用中起什么作用。hHusl 1异二聚体和检查点激活。 总的来说，这些研究将揭示识别DNA损伤并激活调节细胞周期停滞和维持基因组完整性的细胞内信号传导途径的新见解。 此外，了解这一途径的分子细节将确定新的靶点，使细胞对治疗性DNA损伤剂（如电离辐射和抗肿瘤化疗药物）敏感。

Cells continuously experience DNA damage caused by replication errors, metabolically induced oxidative damage, and exogenous mutagens. DNA damage triggers orchestrated cellular responses that include mobilization of repair machinery and activation of cell cycle checkpoints, which arrest cells in G1 and G2/M and slow progression through S phase. Genetic and pharmacologic data demonstrate that in organisms as diverse as yeast and humans, disruption of checkpoint function leads to genetic instability, which correlates with acquisition of oncogenic mutations in mammals. Genetic studies in yeast show that inactivation of checkpoint genes prevents DNA damage-induced cell cycle arrest and sensitizes the yeast to genotoxins. Although many of the yeast checkpoint genes have been identified, our understanding of DNA damage-induced checkpoint activation in mammals has lagged behind. To identify potential regulators of the mammalian DNA- damage response, we cloned hRad1, hRad9, and hHus1, which are human homologs of the S. pombe checkpoint genes, rad1, rad9, and hus1, and we initiated a study to examine the biochemical and cellular functions of these proteins in mammals. Our results demonstrate that these proteins form a complex in human cells. Two members of the complex, hRad9 and hRad1, are phosphorylated and associated with chromatin in response to DNA damage, thus demonstrating that these proteins form a damage-responsive checkpoint complex. Moreover, we provide new data demonstrating that DNA damage provokes a redistribution of hRad9 into nuclear foci. We now propose to extend these findings and define the biochemical functions of hRad9 and identify its roles in cellular checkpoint responses. The Specific Aims of the project are to: 1) determine whether nuclear retention of hRad9 after DNA damage reflects a biochemical sensing mechanism for damaged DNA; 2) analyze the role of hRad9 in checkpoint signaling activation and cell cycle arrest following DNA damage; 3) identify the basal and DNA damage-induced hRad9 phosphorylation sites and determine what role(s) they play in mediating interactions with the hRad1/hHus1 1 heterodimer and in checkpoint activation. Collectively, these studies will reveal novel insights into the pathways that recognize DNA damage and activate intracellular signaling pathways that regulate cell cycle arrest and maintain genome integrity. Additionally, understanding the molecular details of this pathway will identify novel targets to sensitize cells to therapeutic DNA-damaging agents, such as ionizing radiation and anti-tumor chemotherapeutics.