In the yeast Saccharomyces cerevisiae, recombination plays a central role in the repair of DNA double-strand breaks (DSB). Research on X-ray induced DSB and repair in yeast will be continued, making use of the well characterized radiation- sensitive (rad) mutants that we have previously studied. Several different experimental approaches are proposed which all address basic aspects of recombinational repair. Recent pulsed-field gel electrophoresis (PFGE) techniques (Mortimer et al., 1991) will be used to investigate the molecular basis of recombinational repair of DSB. As described earlier, diploid yeast strains that contain one circular and one linear derivative of chromosome III can be used in combination with PFGE to measure the frequency of DSB, and to separately assay molecules that have undergone sister-chromatid exchange (SCE) and those that are products of recombination between homologous chromosomes. Thus, the relative importance in repair of SCE versus interhomolog recombination can be assessed. Several other significant questions about repair will be addressed, for example whether DSB can be repaired by non-recombinational mechanisms in yeast and whether one unrepaired DSB corresponds to a lethal event. The molecular phenotypes of wild-type and rad mutant strains will be characterized using PFGE and molecular endpoints will be related to biological events, including survival, recombination and chromosome loss. We also hope to develop further assays for SCE and for single-strand nicks in yeast using circular chromosomes. In related studies, PFGE will be used to characterize chromosomal aberrations induced by X-rays in yeast. There are extensive studies of such aberrations in higher eukaryotes, but few in lower eukaryotes because of the difficulty of observing aberrations cytologically. PFGE now enables us to detect these aberrations, including translocations and large deletions, and available rad mutants will be used to ask whether recombinational repair plays a role in their formation. The frequency of various types of aberration versus X-ray dose will be determined, both for wild-type strains and for strains mutant in each of the two major types of X-ray repair in yeast. If recombinational repair is important in the formation of aberrations, few such aberrations are expected in mutants blocked in this process. The molecular analysis of genes required for recombinational repair will be continued. The DNA sequencing of genes we have cloned will be completed by finishing the sequencing of the RAD51 and RAD55 genes. We will also focus on the regulation of the RAD51 and RAD54 genes and on obtaining the RAD54 protein. RAD51 and RAD54 possess a common upstream sequence and it will be determined if this is required for their observed induction by DNA damage. These genes will also be tested for cell-cycle regulation. Antibodies against part of the RAD54 protein will be affinity purified and used to assay induced and constitutive levels of this protein, to study its intracellular localization and to purify the intact protein.
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