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theses

Defects in DNA repair are associated with a variety of human diseases, but the pathways that regulate DNA repair are poorly understood. Protein modification by SUMO remains one of the most elusive mechanisms, even though it is known to be important for genome stability. Sgs1, a RecQ DNA helicase in S. cerevisiae, is important for DNA repair, and genetic screens have shown that the heterodimeric Slx5-Slx8 SUMO-targeted Ub ligase (STUbL) is required for cell viability in its absence. Another factor that genetically connects SUMO to DNA repair is the metalloprotease Wss1. The growth of a wss1Δ sgs1Δ double mutant is reduced compared to either single mutant. Taking a candidate-gene over-expression (OE) approach, I made the unexpected finding that SLX5 over-expression is lethal to cells lacking WSS1. Surprisingly, this lethality did not require its partner protein Slx8, which suggested that this was a novel Slx5 activity. Consistent with this lethality, was also observed when STUbLs such as yeast ULS1 or mammalian RNF4 were expressed in these cells. Further, structure-function analysis showed that lethality due to SLX5 OE required only Slx5’s SUMO interaction Motif’s (SIMs), not its RING domain. Kinetic analysis indicated that SLX5 OE slows the growth of wild-type cells. In contrast, SLX5 OE permanently arrests the growth of wss1∆ cells. While investigating the cause of this lethality, I found that expression of Slx5 SIMs resulted in the accumulation of high molecular weight SUMO conjugates in both wild-type and mutant cells. Interestingly, subsequent repression of Slx5 led to a reduction in the levels of induced poly-SUMO chains and this turnover was more rapid in wild-type than in wss1∆ cells. The fact that the accumulation of poly-SUMO chains required the Ubc9 SUMO conjugating enzyme, but not the Ulp1 or Ulp2 isopeptidases, supports the hypothesis that the SIMs actively promote chain synthesis. Finally, I found that over-expression of SUMO counteracts the lethality of SLX5 OE. This suggests that wss1∆ cells may suffer lethal damage due to a shortage of monomeric SUMO.

ETD_6066

M.S.

Includes bibliographical references

by Letzibeth Mendez-Rivera

doi:10.7282/T3H41T47

ETD graduate

The author owns the copyright to this work.