RADIATION INDUCTION OF GENOMIC REARRANGEMENTS IN YEAST

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DNA damage generates recombinogenic double-strand breaks, induces the expression of damage-inducible genes (DIN genes), and arrests the cell cycle at well defined cell-cycle checkpoints. The presence of genomic rearrangements in leukemias and lymphomas, and the correlation between exposure to ionizing radiation and the elevated frequencies of acute leukemia, require a better understanding of how recombinogenic agents can stimulate interchromosomal rearrangements. The general goal is to understand how recombinogenic lesions are repaired so that genomic integrity is maintained. This proposal studies damage-inducible responses in Saccharomyces cerevisae (yeast) by first focusing on the genetic regulation of damage-induced mitotic recombination occurring between dispersed repeated DNA sequences (ectopic recombination). The results obtained in these studies may support four important hypotheses concerning genomic stability in all organisms: 1) that elevated levels of recombinases are correlated with the increased incidence of mitotic rearrangements, 2) that mismatch repair systems are important in aborting recombinational intermediates between divergent sequences and recombinogens may circumvent this control, 3) that topoisomerases and helicases are important in channelling recombinational intermediates into a pathway that does not generate chromosomal rearrangements, and 4) that cell cycle arrest at defined cell cycle checkpoints is necessary to allow these mechanisms to act. The first aim uses the his3 recombinational substrates to quantitate effects of mutations in radiation repair, mismatch repair, topoisomerases, damage-induced sister chromatid recombination and cell cycle control on the rates of spontaneous and damage-induced interchromosomal recombination. The second aim determines whether enhanced expression of radiation-inducible genes, including the RAD51 gene, is correlated with elevated recombination. The third aim is to construct recombinational substrates so that the frequencies of ectopic recombination occurring between any two identical DNA repeats can be quantitated; these substrates will be placed on non-homologous chromosomes so that translocations can be measured. Repeated sequences that will be studied include delta elements occurring at the end of the yeast retrotransposon Ty1 and Alu sequences approximately 300 bp sequence occurring once every 6 kb on average in human DNA cloned on yeast artificial chromosomes (YACs). Recombination assays will then determine whether mitotic, ectopic recombination between non-identical repeats can be stimulated by DNA damaging agents. The fourth aim is to determine whether mutants in the mismatch repair pathways exhibit higher frequencies of recombination between these sequences and whether over-expression of the RAD51 recombinase will enhance recombination between these repeats. Results from this study will aid in understanding the genetic control of genomic stability in yeast, facilitate the maintenance of foreign DNA introduced in yeast on YACs, and provide insights into the recombinogenicity of DNA damaging agents in eukaryotic systems. In addition, the novel recombinational substrates designed for yeast will be of benefit to geneticists interested in creating similar recombinational substrates for higher eukaryotes.