P53 MEDIATED G2/M CHECKPOINT CONTROL

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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.