An investigation into the function of sumoylation in genomic stability in schizosaccharomyces pombe

Mercer, Brenda (2013) An investigation into the function of sumoylation in genomic stability in schizosaccharomyces pombe. Doctoral thesis (PhD), University of Sussex.

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Sumoylation is an essential posttranslational modification involved in many cellular
processes such as DNA replication, chromosomal stability, cytokinesis, DNA damage
responses and many others. The process of sumoylation is conserved in all eukaryotic
organisms. This study involves the analysis of various aspects of sumoylation in the
unicellular model organism Schizosaccharomyces pombe.

The first part of this study is concerned with elucidating the functional and structural
importance of a SUMO-like domain (SLD) and a putative SUMO-binding domain (SBM)
present in the essential protein Rad60. Biochemical and genetical analysis reveals that
SLD2 is required for the DNA damage response function of Rad60 but that the putative
SBM3 is a key structural feature of the hydrophobic core of SLD2 and therefore unlikely
to function as a SUMO-interacting motif. As Rad60 interacts with the SUMO E3 ligase
Pli1, which facilitates overall sumoylation and SUMO chain formation, further analysis
was undertaken to identify the function of SUMO chain formation and the function of
Pli1 in maintaining chromosomal stability. A SUMO chain mutant, Pmt3-K14R; K30R,
was characterized and shown to be sensitive to the DNA replication inhibitor
hydroxyurea. Analysis of the pli1 null mutant reveals that Pli1 dependent sumoylation
has multiple functions at the centromeric repetitive sequences as the mutant displays
increased gene conversion at centromeric regions and increased loss of an artificial
minichromosome rates compared to a wild type strain.

The second part of this study is concerned with identifying specific modified lysine
residues in the sumoylation pathway components Fub2, Hus5 and Nse2 and the target
proteins Rtf2 and PCNA. After identification of in vitro sumoylation sites, an analysis of
the sumoylation of the SUMO conjugating enzyme Hus5 and the sumoylation of the Rtf2
protein is carried out. In vivo and genetical analysis of the hus5-K50R mutant suggests
that the sumoylation of the conjugating enzyme is required for maintaining the
homeostasis of the pathway and is essential for cell viability when the homologous
recombination machinery is impaired. Sumoylation of Rtf2 protein is required for the
response to the DNA alkylating agent MMS and, like the sumoylation of Hus5, is
essential for cell viability in homologous recombination mutant backgrounds.

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: 28 Nov 2013 07:38
Last Modified: 17 Sep 2015 13:13

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