Recombinational repair, including sister-chromatid exchange, is one of the three major repair pathways in yeast, and is crucial for the repair of x-ray induced double-strand breaks and is also involved in the repair of many types of chemical damage, including DNA cross-links. Recombinational repair is probably also utilized in mammalian cells, and recently human genes have been isolated which are homologs of the three yeast genes we will be studying. Since several repair genes have been strongly implicated in carcinogenesis, a better understanding of how repair genes function should give us important information which will have implications for both carcinogenesis and how cells deal with exposure to environmental carcinogens. Investigation of recombinational repair in the yeast S. cerevisiae, emphasizing the three genes (RAD51, 52 and 54) most necessary for this process, will be continued.
The specific aims relate to the importance of different domains of the RAD54 protein, a member of a large protein family including the protein mutated in Cockayne's syndrome B, the interactions between proteins involved in recombinational repair, and the order of function of genes in this pathway. The proposed experiments will give us additional information about this important repair pathway. The RAD54 gene is necessary for recombinational repair, but it is not widely studied. Molecular and biochemical studies will be initiated to examine interesting regions encoding a putative nucleotide-binding domain, potential DNA helicase domains, and a probable zinc finger. Site specific mutagenesis using PCR primers will be used to introduce mutations into potential domains. These mutations will be tested for their effect on repair. Low fidelity PCR will be used to isolate random rad54 mutations, which will be screened for temperature-conditional and leaky alleles. Interesting alleles will be sequenced and used in other experiments. In conjunction with these molecular experiments, we will also isolate the RAD54 protein and a truncated form containing the zinc finger region. We will attempt to isolate RAD54 from E. coli, but use overexpression in yeast if necessary. This protein will be characterized for several activities, including ATPase activity, zinc binding, binding to strand breaks, and helicase activity. Some mutant RAD54 proteins will also be characterized. The RAD52 and RAD51 proteins have recently been reported to interact. -We plan to determine if this protein interaction is essential for recombinational repair, by further characterizing our rad52-20 allele which may be defective in this interaction. To test if the rad52-20 protein has weakened interaction with RAD51, we will use the molecular two-hybrid system. Studies will also be initiated to determine if the RAD54 protein interacts with other proteins, using both genetic reversion of conditional rad54 alleles and the two-hybrid system. Experiments are proposed to examine the functional order of the RAD51, 52 and 54 genes, using temperature shift experiments in conjunction with temperature-sensitive (t.s.) and cold-sensitive (c.s.) alleles. We plan to determine if RAD54 functions before or after RAD51 and RAD52, using our recently isolated c.s. and t.s. rad51 alleles in combination with the t.s. rad54-3 allele and new t.s. rad52 alleles, isolated by Kaytor and Livingston.
Showing the most recent 10 out of 27 publications
