All ETDs from UAB

Advisory Committee Chair

Mary-Ann Bjornsti

Advisory Committee Members

Stephen Barnes

David M Bedwell

Eric J Sorscher

Robert C A M Van Waardenburg

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

This dissertation seeks to uncover the impact of a post-translational modification, the small ubiquitin-related modifier (SUMO), on the cell response to DNA damage. Covalent modification of cellular proteins by SUMO occurs through an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and a ligase (E3). Eukaryotic cells possess a single E1 and a single E2 for SUMO conjugation. The E2 for SUMO, Ubc9, is able to recognize and modify substrates on its own, although substrate recognition and modification are enhanced in the presence of the E3. Two independent genetic screens carried out in the budding yeast Saccharomyces cerevisiae identified a mutant expressing Ubc9 with a proline to leucine substitution at position 123 in its amino acid sequence (Ubc9P123L). This temperature-sensitive mutant was viable in the presence of other cellular stresses, but slow-growing and hypersensitive to DNA damage at 36°C. In this mutant, the essential, viability-maintenance function of Ubc9 was dissociated from its function in protecting the cell from DNA damage. In this dissertation, several approaches were employed to study Ubc9P123L in order to understand the basis for this dissociation of function. NMR studies of protein structure yielded evidence suggesting that Ubc9P123L is impaired in its ability to recognize and modify lysine residues in extended regions, which was corroborated in vitro and in yeast cells through studies of other proline 123 (Pro123) substituents in yeast Ubc9. These studies provide the first evidence that the different linkages SUMO is able to form may have distinct consequences for the cell. Finally, a proteomic approach was attempted in order to identify proteins that showed differences in SUMO modification status in yeast cells expressing Ubc9P123L versus wild-type cells. Although those studies remain incomplete, they have led to the development of yeast strains from which substrate-level and site-specific information about cellular SUMO modification can be obtained in a single mass spectrometry experiment.

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