This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Superoxide dismutase is a metalloenzyme that catalyzes the conversion of two molecules of superoxide anions to hydrogen peroxide and water and thus plays a key role in protecting aerobic cells against oxidative stress. We have previously shown that loss of Cu, Zn-dependent superoxide dismutase, SOD1, or its copper chaperone, LYS7, confers oxygen-dependent sensitivity to replication arrest and DNA damaging agents and reduced induction of Hug1p and the MEC1 pathway effector Rnr3p in S. cerevisiae. The HU sensitivity of sod1 trains is suppressed by overexpression of TKL1, a transketolate that generates NADPH, which balances redox in the cells and is required for the ribonucleotide reductase activity. We are testing the hypothesis that SOD1 is required for signaling through the MEC1 pathway, maintenance for optimal dNTP pools, cell cycle progression and recovery from DNA damage. We have observed that the sod1 trains show a delay in progression through G1 phase of the cell cycle. These results are consistent with phenotypic consequences of low dNTP pools in the sod1 trains. In order to gain further insight into the role of Sod1p, we conducted a synthetic genome analysis (SGA) of sod1 ith H 4,400 haploid deletion strains. Analysis and confirmation of the SGA data has established that sod1 trains exhibit a synthetic lethality or synthetic sick phenotype when combined with deletions of genes required for DNA repair, synthesis and metabolism. We propose to do a two-hybrid screen with Sod1p to gain further insights into the molecular role of Sod1p. We propose that the DNA synthesis/repair pathways are essential for haploid growth in the sod1? strains, which are defective in the MEC1 pathway response and their inability to cope with increased endogenous DNA damage due to excessive superoxide ions.
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