ANALYSIS OF A DNA DAMAGE INDUCIBLE CHECKPOINT COMPLEX

细胞不断经历复制错误、代谢诱导的氧化损伤和外源诱变剂造成的DNA损伤。DNA损伤会触发精心安排的细胞反应，包括动员修复机制和激活细胞周期检查点，使细胞停滞在G1和G2/M期，并减缓细胞在S期的进展。遗传学和药理学数据表明，在像酵母和人类这样的生物中，检查点功能的破坏会导致遗传不稳定，这与哺乳动物获得致癌突变有关。酵母中的遗传学研究表明，检查点基因的失活可以防止DNA损伤导致的细胞周期停滞，并使酵母对遗传毒素敏感。虽然许多酵母检查点基因已经被鉴定出来，但我们对DNA损伤诱导的哺乳动物检查点激活的了解还比较滞后。为了确定哺乳动物DNA损伤反应的潜在调控因子，我们克隆了庞贝链霉菌检查点基因Rad1、rad9和hHus1的人类同源基因hRad1、hRad9和hHus1，并启动了一项研究，以检测这些蛋白在哺乳动物中的生化和细胞功能。我们的结果表明，这些蛋白质在人类细胞中形成了一个复合体。该复合体的两个成员hRad9和hRad1被磷酸化，并与染色质结合以响应DNA损伤，从而表明这些蛋白形成了一个损伤响应检查点复合体。此外，我们还提供了新的数据，表明DNA损伤会导致hRad9重新分布到核内。我们现在建议扩展这些发现，定义hRad9的生化功能，并确定它在细胞检查点反应中的作用。该项目的具体目标是：1)确定DNA损伤后hRad9的核滞留是否反映了对损伤的DNA的生化传感机制；2)分析hRad9在DNA损伤后的检查点信号激活和细胞周期停滞中的作用；3)确定DNA损伤诱导的基础和DNA损伤诱导的hRad9磷酸化位点，并确定它们在介导与hRad1/hHus1异源二聚体的相互作用和检查点激活中扮演的角色(S)。总的来说，这些研究将揭示识别DNA损伤和激活细胞内信号通路的新见解，这些信号通路调节细胞周期停滞和维持基因组完整性。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损伤剂敏感，如电离辐射和抗肿瘤化疗药物。

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.