DNA damage provokes cohesin sumoylation in Saccharomyces cerevisiae
Author(s)
McAleenan, Alexandra Susan Jane
Type
Thesis
Abstract
Cohesin tethers sister chromatids together from their creation during S-phase until
their separation at the metaphase to anaphase transition. Cohesion is essential for correct
segregation and therefore genomic stability. Cohesin is also involved the regulation of
transcription and in DNA repair. Cohesin affects these processes by its ability to hold two
pieces of DNA together.
Cohesin is subject to a variety of post-translational modifications. In this study,
the modification of cohesin, more specifically the Scc1 subunit, by Small ubiquitin-like
modifier (SUMO) was analysed. It was found that cohesin is sumoylated during an
unperturbed cell cycle, but is hyper-modified after DNA damage. To determine the
function Scc1 modification by SUMO, attempts were made to identify the modified
residues. Mass spectrometry techniques for SUMO site identification were developed in
parallel to site-directed mutagenesis studies.
Potential sumoylation sites were identified on Scc1, but when these were mutated
there was little effect on either viability or the sumoylation pattern obtained. Mutants
were then constructed in which only one lysine was present. Surprisingly, several of these
were viable, not sensitive to methyl methanesulfonate and were not sumoylated,
suggesting that either sumoylation of Scc1 is not essential and not required after DNA
damage, or that sumoylation of the rest of the cohesin complex can compensate.
To investigate this, Scc1 fusions with the SUMO E2 ligase, Ubc9, and a SUMO
isopeptidase, Ulp1, to mimic constitutively sumoylated and unsumoylatable cohesin
respectively, were constructed. Unexpectedly, it was found that both fusions relocalized to
a double strand break, and that the Ulp1 fusion was recruited to a greater extent. Data
from our laboratory suggests that the Ubc9 fusion is not competent for cohesion after
damage, although it is sufficient for viability. Sumoylation may therefore be involved in
cohesin unloading or turnover, which might be required after DNA damage.
their separation at the metaphase to anaphase transition. Cohesion is essential for correct
segregation and therefore genomic stability. Cohesin is also involved the regulation of
transcription and in DNA repair. Cohesin affects these processes by its ability to hold two
pieces of DNA together.
Cohesin is subject to a variety of post-translational modifications. In this study,
the modification of cohesin, more specifically the Scc1 subunit, by Small ubiquitin-like
modifier (SUMO) was analysed. It was found that cohesin is sumoylated during an
unperturbed cell cycle, but is hyper-modified after DNA damage. To determine the
function Scc1 modification by SUMO, attempts were made to identify the modified
residues. Mass spectrometry techniques for SUMO site identification were developed in
parallel to site-directed mutagenesis studies.
Potential sumoylation sites were identified on Scc1, but when these were mutated
there was little effect on either viability or the sumoylation pattern obtained. Mutants
were then constructed in which only one lysine was present. Surprisingly, several of these
were viable, not sensitive to methyl methanesulfonate and were not sumoylated,
suggesting that either sumoylation of Scc1 is not essential and not required after DNA
damage, or that sumoylation of the rest of the cohesin complex can compensate.
To investigate this, Scc1 fusions with the SUMO E2 ligase, Ubc9, and a SUMO
isopeptidase, Ulp1, to mimic constitutively sumoylated and unsumoylatable cohesin
respectively, were constructed. Unexpectedly, it was found that both fusions relocalized to
a double strand break, and that the Ulp1 fusion was recruited to a greater extent. Data
from our laboratory suggests that the Ubc9 fusion is not competent for cohesion after
damage, although it is sufficient for viability. Sumoylation may therefore be involved in
cohesin unloading or turnover, which might be required after DNA damage.
Date Issued
2010
Date Awarded
2010-05
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Aragon, Luis
Creator
McAleenan, Alexandra Susan Jane
Publisher Department
MRC Clinical Sciences Centre
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)