From the studies of DNA repair and recombination using various organisms, it is apparent that nucleases play a major role in both of these processes. In S. cerevisiae the RAD52 gene is essential for the repair of DNA double-strand breaks, mitotic recombination and for the successful completion of meiosis. A 72 kd nuclease had been identified and subsequently purified. In rad52 mutants, levels of yNucR are greatly decreased suggesting that this nuclease is under the control of RAD52. Using a lambda gt11 expression library and an antibody which immunoprecipitates yNucR, the gene encoding yNucR has been cloned. Subsequently, a clone encoding the entire yNucR has been isolated from a YEp2l3 yeast genomic library. Using lacZ fusion analysis, the direction of transcription of yNucR has been determined. With the yNucR::lacZ fusion, the levels of the fusion protein has been determined during meiotic growth and in response to ionizing radiation. During meiosis, yNucR::lacZ increases approximately 2-3 fold and prior to the commitment to recombination. However, the levels of fusion protein slightly decrease in response to ionizing radiation. Western bolts of wild-type yNucR in cells previously irradiated indicate that yNucR greatly decreases after irradiation (by two hours post-irradiation cross-reacting material disappears). These results suggest a proteolytic cleavage of yNucR or high turnover of the protein in response to ionizing radiation. Using the clone for yNucR, we have begun gene disruption studies of the gene. Initially, our studies indicated that gene disruption of yNucR leads to lethality. However, this lethality appears to be strain specific. Chromosome blots using the cloned yNucR indicates that this clone hybridizes to both chromosome XI and VI. Therefore, there may exist different functional copies of yNucR and the number of copies may be strain specific. Further molecular and genetic analysis is currently underway to determine the number of functional copies of yNucR encoded in the yeast genome.
NIH ExPorter
NIH ExPorter