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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES021016-09
Application #
3876835
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Andres, Sara N; Appel, C Denise; Westmoreland, James W et al. (2015) Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22:158-66
Ma, Wenjian; Westmoreland, Jim W; Gordenin, Dmitry A et al. (2011) Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease. PLoS Genet 7:e1002059
Nakai, Wataru; Westmoreland, Jim; Yeh, Elaine et al. (2011) Chromosome integrity at a double-strand break requires exonuclease 1 and MRX. DNA Repair (Amst) 10:102-10
Argueso, Juan Lucas; Westmoreland, James; Mieczkowski, Piotr A et al. (2008) Double-strand breaks associated with repetitive DNA can reshape the genome. Proc Natl Acad Sci U S A 105:11845-50
Chen, Ling; Trujillo, Kelly M; Van Komen, Stephen et al. (2005) Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast. J Biol Chem 280:2620-7
Resnick, Michael A (2005) Reduced replication: a call to ARMS. Cell 120:569-70
Lewis, L Kevin; Karthikeyan, G; Cassiano, Jared et al. (2005) Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair. Nucleic Acids Res 33:4928-39
Lewis, L Kevin; Lobachev, Kirill; Westmoreland, James W et al. (2005) Use of a restriction endonuclease cytotoxicity assay to identify inducible GAL1 promoter variants with reduced basal activity. Gene 363:183-92
Lewis, L Kevin; Storici, Francesca; Van Komen, Stephen et al. (2004) Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells. Genetics 166:1701-13
Lobachev, Kirill; Vitriol, Eric; Stemple, Jennifer et al. (2004) Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex. Curr Biol 14:2107-12

Showing the most recent 10 out of 11 publications