CELL CYCLE RESPONSE TO RADIATION DAMAGE IN YEAST

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The overall goal is to elucidate the mechanisms of cell cycle arrest in the yeast Saccharomyces cerevisiae following treatment with UV and ionizing radiation, with special emphasis on the nature of the checkpoint-activating DNA damage and the mechanism of its recognition. Eukaryotic cells have the capability to reversibly delay cell cycle progression in response to radiation damage at discrete transition points termed checkpoints. It is assumed that checkpoint arrest can provide time for DNA repair in order to avoid irreversible fixation of damage in the form of mutations or chromosome aberrations. Thus, the elucidation of checkpoint mechanisms and an analysis of their inactivation in cancer cells is of importance for understanding the process of cellular transformation and the phenotype of genetically unstable cancer cells. The genetically highly amenable yeast Saccharomyces cerevisiae has already served successfully as a model organism to establish the concept of cell cycle checkpoints. Various mutants are known to be defective in 61, 62 and in 5-phase checkpoints. It is proposed to analyze the mechanisms of radiation-induced cell cycle arrest in yeast by various complementary approaches. The specific aims are as follows: 1.Characterization of activities involved in DNA interactions required for the recognition of checkpoint-activating damage. This includes the purification and characterization of Radl7, a putative 3'>5' exonuclease involved in checkpoint arrest and a potential yeast homolog of the human tumor suppressor and checkpoint determinant p53. Additionally, an analysis of protein binding to candidate target DNA substrates will provide information on the nature of the cell-cycle arrest- triggering DNA structures. 2.A screen for proteins that interact with the known checkpoint controlling gene products Rad17-and Mec1. The methodology includes the well-established yeast two-hybrid system, a selection for multi-copy suppressor genes and expression of His-tagged checkpoint-controlling proteins. Genes for interacting proteins will be inactivated and the phenotype of the resulting mutants will be analyzed. 3.Selection and characterization of novel yeast mutants defective in G1 arrest. 4.A screen for human homologs of checkpoint-controlling yeast genes by PCR-based strategies or by functional complementation of yeast mutants with human cDNA libraries. Additionally, potential functional homology between yeast Rad17 and human p53 will be explored in complementation studies.