This project will study the effects of mutations in genes that regulate the onset of mitosis. Mitosis is begun by activation of a factor known as MPF for mitosis-promoting factor. MPF activity is controlled by phosphate content, subunit structure, location within cells and the status of chromosomes. A surveillance system known as the G2 checkpoint monitors the structure of chromosomes and can inhibit MPF when damage is sensed. After the damage is repaired, the inhibition of MPF is removed and progression of cells to mitosis resumes. Gene mutations that reduce G2 checkpoint function are the focus of this project. Expression of mutant forms of the cyclin-dependent kinase, Cdk1, the catalytic subunit of MPF, or the phosphatase, Cdc25C, that activates MPF, and overexpression of the MPF regulatory subunit, cyclin B1 reduced G2 delay in HeLa cells with damaged DNA, implicating these genes as functional components of the G2 checkpoint in a line of cancer cells. These genes will be induced in diploid human fibroblasts to establish their roles in G2 checkpoint function in normal cells. An enzyme known as DNA topoisomerase II detangles intertwined chromatids after DNA replication. A brief interval of inhibition of topoisomerase II in G2 cells was found to delay the onset of mitosis for several hours, as though a checkpoint had been activated. The hypothesis that chromatid catenation status is monitored by the G2 checkpoint will be tested by determining whether human cell lines with defects in G2 checkpoint genes cannot delay growth in G2 when chromatid detangling is inhibited. Human fibroblasts that bypass the replicative senescence checkpoint due to expression of HPV16E6 oncoprotein display progressive chromosomal destabilization and loss of G2 checkpoint function. Studies will determine whether expression of telomerase to stabilize telomeres preserves G2 checkpoint function in E6-expressing cells. To determine whether the loss of G2 checkpoint function in immortal fibroblasts is a dominant or recessive trait, checkpoint- proficient cells will be fused with checkpoint-defective cells and the function of the checkpoint in cell hybrids will be quantified. This project will define inputs to the G2 checkpoint and identify mechanisms of inactivation in human fibroblasts.
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