The response to DNA damage of organisms from E. coli to humans is marked by two major features, arrest of cell cycle progression, and induction of genes required for DNA repair. In eukaryotes, several gene products (most notably the ESR1/MEC1, RAD3, MEI41, and ATM kinases) are conserved and required for cell cycle arrest after DNA damage. Mutations in ATM or other checkpoint genes including p53 result in increased predisposition to malignancy and alteration in the response to radiation therapy. A novel human cDNA, CHES1 (Checkpoint Suppressor 1) encoding a member of the fork head/Winged Helix family which can reconstitute a MEC1- independent checkpoint when expressed in yeast has been recently isolated. Analysis of the activity of CHES1 is consistent with activation of a novel alternative checkpoint pathway. This proposal builds on this observation to define the genes required for this alternative checkpoint pathway in yeast and determine the activity of both endogenous and exogenous CHES1 in mammalian cells. The experimental aims include a systematic genetic screen to identify mutants of S. cerevisiae which have lost the alternative pathway. In parallel, direct isolation of both yeast and human genes which interact with CHES1 will define which gene product in the alternative pathway is being activated by CHES1. The mammalian homologs of the genes identified in these aims will be characterized for their ability to modulate the response to DNA damage of mammalian cells. Given the ability of CHES1 to suppress the DNA damage sensitivity of yeast deleted for MEC1 direct determination of whether exogenous expression of CHES1 will suppress the checkpoint defect in cells from patients with ataxia telangiectasia (AT) will be performed. This will be accomplished by mass infection of cultures with an adenoviral construct expressing CHES1. These latter results will demonstrate whether activation of an alternative checkpoint pathway might be used therapeutically for patients with AT or to alter the resistance of tumors to radiation and other DNA-damaging agents.
