P53 MEDIATED G2/M CHECKPOINT CONTROL

研究表明，细胞在反应中阻止细胞周期进程 DNA损伤，以便有时间进行修复，从而避免有害的 突变和染色体损伤的影响。哺乳动物细胞中的阻滞 通常依赖于p53肿瘤抑制因子的蛋白产物 基因 p53基因在不同类型的人类中经常发生突变。 癌症，并认为肿瘤发生的过程是 与p53在介导细胞周期中的功能密切相关 检查点控制 暴露于DNA损伤的细胞在G1期和G2期均停滞。 细胞周期的G2期。 G1期阻滞已被证明是 部分由p53依赖的基因转录激活介导 编码p21 Cip 1/Waf 1/SD 11蛋白、细胞周期蛋白依赖性激酶 抑制剂. 研究还表明，p53可能参与DNA 损伤诱导的G2期阻滞;然而，p53介导的G2期阻滞的机制 逮捕并不容易理解。 我们最近发现了一个新的基因，Wip 1， 在p53依赖性细胞中， 方式 Wip 1基因编码的p60 wip 1蛋白具有同源性 和2C型蛋白磷酸酶的生化特性。 初步数据显示p60 wip 1的异位表达 在G2期阻滞细胞周期进程， 与激酶活性的急剧下降有关， cdc 2/cyclin B复合物。 我们认为p60 wip 1是一个下游的 p53介导的G2检查点控制的效应子。 的具体目标 这是以下内容。一号目标 从生物化学的角度 表征p60 wip 1的G2检查点的蛋白组分 在G2期阻滞时相互作用。 目标二 以确定是否 p60 wip 1与G2期阻滞细胞中的未知蛋白相互作用， 鉴定、克隆和表征这些蛋白质。 目标三到 确定Wip 1基因的改变是否会影响 细胞对电离辐射和其他DNA损伤的敏感性 剂.

Studies have shown that cells arrest cell cycle progression in response to DNA damage to allow time for repair, thus avoiding the deleterious affects of mutations and chromosomal damage. Arrest in mammalian cells is often dependent on the protein product of the p53 tumor suppressor gene. The p53 gene is frequently mutated in diverse types of human cancers and it is thought that the process of tumorigenesis is intimately related to the functionality of p53 in mediating cell cycle checkpoint control. Cells exposed to DNA damage arrest in both G1- and G2-phase of the cell cycle. G1-phase arrest has been shown to be mediated in part by p53-dependent transcriptional activation of the gene which encodes p21Cip1/Waf1/SDl1 protein, cyclin-dependent kinase inhibitor. Studies have also suggested that p53 may be involved in DNA damage-induced G2 arrest; however, the mechanism(s) of p53-mediated G2 arrest are not well understood. We have recently identified a novel gene, Wip1 that is transcriptionally-activated in response to DNA damage in a p53-dependent manner. The Wip1 gene encodes p60wip1 protein which exhibits homology and biochemical characteristics of a type 2C protein phosphatase. Preliminary data is presented showing that ectopic expression of p60wip1 arrests cell cycle progression in G2-phase and that the arrest is associated with a dramatic decrease in the kinase activity of cdc2/cyclin B complexes. We propose that p60wip1 is a downstream effector of p53-mediated G2 checkpoint control. The Specific Aims of this are the following. Aim number 1. To identify and biochemically characterize protein components of the G2 checkpoint that p60wip1 interacts with during G2 arrest. Aim number 2. To determine whether p60wip1 interacts with unknown proteins in G2 arrested cells and to identify, clone, and characterizes these proteins. Aim number 3. To determine whether alterations in the Wip1 gene affect the relative sensitivity of cells to ionizing radiation and other DNA-damaging agents.