An investigation into the function of the SUMOylation of Nse2 and PCNA in S. pombe

Small, Lauren (2014) An investigation into the function of the SUMOylation of Nse2 and PCNA in S. pombe. Doctoral thesis (PhD), University of Sussex.

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Small ubiquitin like modifier (SUMO) is post-translationally attached to target proteins, forming a covalent bond between its C-terminal glycine and one or more lysine residues on the target protein. SUMO modification of target proteins can affect protein-protein interactions, protein activity, localistation and stability. This study set out to develop an efficient in vitro SUMOylation system to enable the identification of target lysine residues in S. pombe proteins by mass spectrometry. This involved incorporating a trypsin cleavage site adjacent to the SUMO di-glycine motif to improve peptide coverage during mass spectrometry. Several SUMOylated target proteins were identified here, including the E2 SUMO conjugating enzyme Hus5, the E3 SUMO ligase Nse2 and PCNA.
The second part of this study focused on the characterisation of unSUMOylatable E3 SUMO ligase nse2 mutants. Integration of lysine to arginine mutations into the genome did not result in any mutant phenotypes and a function for auto-SUMOylation of Nse2 was not identified. During this study, human patients with mutations in the nse2 gene were reported and the equivalent mutations were integrated into the S. pombe nse2 gene to investigate the effect of the mutations.
The final part of this work involved the analysis of the SUMOylation of S. pombe PCNA. Using the in vitro system, four target lysine residues for SUMO were identified. SUMOylation of PCNA was also observed in vivo following pull-down studies and 2D gel analysis of wild type and unSUMOylatable mutants. Extensive epistasis analysis was undertaken using these mutants to investigate the role of SUMOylation of S. pombe PCNA.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 04 Feb 2015 11:40
Last Modified: 28 Sep 2015 13:04

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