Ubiquitin conjugating enzyme 1 (Ubc1) is a member of the E2 family of enzymes that conjugates ubiquitin to damaged proteins destined for degradation by the ubiquitin proteasomal system. It is necessary for stress tolerance and is essential for cell survival in Saccharomyces cerevisiae. Ubc1 has five serine residues that are potential substrates for phosphorylation by kinases. However, no data are available to indicate that Ubc1 function or stress tolerance in S. cerevisiae is regulated by serine phosphorylation of Ubc1. We demonstrate that Ubc1 is phosphorylated in serine residue(s). Furthermore, expression of Ubc1 mutants that are 'constitutively phosphorylated' or 'dephosphorylated' in mitogen-activated protein (MAP) kinase serine residues (S97 and S115) affected tolerance to thermal and reductive stress in S. cerevisiae. Specifically, expression of Ubc1S97A and S115D increased thermo-tolerance in both BY4741 and TetO 7 -UBC1ura3Δ cells. Serine phosphorylation of Ubc1 was decreased in BY4741 cells following exposure at 40 C. Tolerance to reductive stress in the same strains correlated with the expression of Ubc1S97A. Ubc1 phosphorylation did not show significant alteration under similar conditions. Both hog1Δ and slt2Δ cells expressing Ubc1S115D and Ubc1S115A were rendered tolerant to thermal and reductive stress respectively. Ubc1 phosphorylation was higher in BY4741 cells compared to hog1Δ cells at 30 C and was significantly reduced in BY4741 cells upon exposure at 40 C. Taken together, the cell survival assays and Ubc1 phosphorylation status in strains and under conditions as described above suggest that tolerance to thermal and reductive stress in S. cerevisiae may be regulated by MAP kinase-mediated phosphorylation of Ubc1S97 and S115.
DNA repair functions are essential for the maintenance of genetic integrity and are regulated in response to both environmental and chemical stressors in mammalian and yeast cells in culture. The inhibitory effect of limited O 2 availability on DNA repair functions in general and on homologous recombination (HR) in particular, correlates with increased chromosomal abnormalities in hypoxic cancer cells. Given the above, we have investigated the effects of CoCl 2 ,--a hypoxia mimetic agent on HR and genetic aberrations in Saccharomyces cerevisiae. Our studies demonstrate that both acute and chronic exposure to CoCl 2 activated HR and increased genetic aberrations in S. cerevisiae D7 cells. At early time points following addition of CoCl 2 to the growth media, cells were briefly arrested in the G1-S boundary concomitant with a transient increase in Rad52-GFP foci formation and induction of low levels of DNA damage. The mode of action of CoCl 2 is thus similar to that of DNA synthesis inhibitors, the later are known to induce HR and cause G1-S arrest. We propose that the activation of HR in the presence of the hypoxia mimetic agent may be attributed to the replication stress and/or DNA damage induced by the stressor.Keywords Saccharomyces cerevisiae Á Hypoxia mimetic Á Homologous recombination Á DNA damage Á G1-S arrest Intracellular DNA double-strand breaks (DSB) generated by exposure to ionizing radiation, chemotherapeutic drugs and reactive oxygen species, as well as during V(D)J recombination and immunoglobulin class switching are repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Failure to repair DSBs results in various chromosomal abnormalities that may ultimately lead to neoplastic transformation [1]. In S. cerevisiae, HR is the major pathway for DNA double strand break repair (DSBR) and is carried out by members of the RAD52 epistasis group that include RAD52, RAD50, RAD51, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2 and requires extensive DNA sequence homology between the DNA molecules involved in recombination. HR is initiated by DSBs followed by 5 0 to 3 0 resection of the ends. The 3 0 ends thus formed are recombinogenic that bind Rad51 in an ATP dependent reaction and invades homologous template to initiate DNA replication by strand displacement. The inhibitory effect of RPA on Rad51 binding to DNA is overcome by Rad52. Broken ends are then joined by ligation leading to the formation of two Holliday junctions that are subsequently resolved to yield either crossover or non-crossover products [2]. DSBR by NHEJ requires little or no sequence homology and is carried out by the MRX complex along with Yku80, Yku70, DNA Pol4 and DNA ligase IV [3]. While DSB repair by HR is error-proof, that by NHEJ is an error-prone process.DNA replication and repair functions are regulated in response to environmental stress conditions in S. cerevisiae. Hypoxic stress results in transient down-regulation in the expression of MCB and SCB regulated genes required
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