This collaborative proposal will be carried out primarily in Uruguay as an extension of NIH grant # RO 1 -CA87381. The main goal to study the interactions between cell cycle checkpoint arrest and DNA strand break repair in the budding yeast Saccharornyces cerevisiae with special emphasis on the checkpoint protein Rad17. Specifically, it is proposed 1. To study DNA double-strand break repair in a rad17 mutant under exclusion of cell cycle progression. This will be accomplished by incubating irradiated diploid strains under non-growth conditions and analyzing the integrity of the chromosomes in pulsed-field agarose gels. Thus, the frequently debated issue will be addressed whether the known radiation sensitivity of checkpoint mutants such as rad17 is solely due to an insufficient time window for repair (due to a defect in cell cycle arrest) or whether these genes play an additional direct role in DNA repair. Double mutant analysis will indicate the repair pathway that is affected by Rad17. 2. To characterize DNA double-strand break repair in a rad17 and a chk1 mutant under conditions that will activate cell cycle checkpoints in normal cells. In this aim, it will be investigated if the failure to arrest at a checkpoint (specifically at the G2/M checkpoint) in growing cells translates into incomplete strand break repair. Thus, the commonly assumed correlation between radiation sensitivity, checkpoint arrest, and repair capacity will be explored. It will be of interest to compare rad17 and the recently isolated chk1 mutant in this respect since a less pronounced defect in G2/M arrest and virtually wild-type sensitivity towards DNA damaging agents was found in the latter mutant. The chromosome profiles of cells of surviving colonies following strand break damage will be analyzed in the rad17 mutant and the pattern of chromosomal aberrations will be studied. 3. To determine the effect of mutant versions of Rad17 in repair under non-growth and growth conditions. In summary, this project addresses the consequences of defective checkpoint control for DNA repair and genetic stability. Genetic instability has been recognized as a key process in the multistep sequence of events leading to human cancer. The project is therefore highly relevant to human health.
