Regulation of the activity of a budding yeast DNA damage repair enzyme Sae2




Fu, Qiong, Ph. D.

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In response to DNA damage, many repair and signaling molecules mobilize rapidly to the sites of DNA double-strand breaks (DSBs). This network of immediate responses is regulated at the level of post-translational modifications to coordinate DNA repair and checkpoint signaling. Here we investigate the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DSB repair. Sae2 is a target of multiple phosphorylation events, which we identify and characterize in vivo in budding yeast. Both cell cycle-dependent and DNA damage-induced phosphorylation of Sae2 are important for the cell survival after DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We find that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by these series of phosphorylation events. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. This analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by post-translational modifications that regulate its solubility and oligomeric state. Budding yeast Ess1 is a phosphorylation-specific prolyl isomerase. Its human homolog Pin1 is found to isomerize CtIP, the human functional ortholog of Sae2, and promote the proteasomal degradation of CtIP. However, I could neither detect any interaction between Ess1 and Sae2, nor observe any change in Sae2 protein level while overexpressing wild-type or mutant Ess1, suggesting Ess1 does not act on Sae2, like Pin1 does on CtIP. The increased DNA damage sensitivity of Ess1 mutants indicates that Ess1 is involved in DNA repair, but not related to Sae2. Since Ess1 plays an important role in transcription termination together with a RNA 3’ end processing factor Pcf11, I overexpressed wild-type Pcf11 and found it significantly increased the DNA damage resistance of either wild-type or H164R mutant Ess1 cells, and also the sae2Δ cells. These results imply that Ess1, Pcf11 and Sae2 might contribute to DNA damage repair through transcription termination, which links transcription termination and DNA damage repair together.




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