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Phosphorylation events surrounding the DNA damage response in "Saccharomyces cerevisiae"

Schleker, Thomas Andreas. Phosphorylation events surrounding the DNA damage response in "Saccharomyces cerevisiae". 2007, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8028

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Abstract

Protein phosphorylation mediated by checkpoint kinases is crucial for the cellular
response to DNA damage. The sensor kinases Mec1 and Tel1 initiate the checkpoint
signaling cascade by directly activating the checkpoint effector kinase Rad53. This
checkpoint pathway, however, is responsive to normal endogenous replication as
well. As a result an S-phase specific threshold for Rad53 activation exists, which
allows cells to tolerate endogenous damage-like structures.
Here we show that Rad53 itself is phosphorylated in a cell-cycle dependent manner
independent of DNA damage signaling (Chapter 2). We propose that this is part of the
cell-cycle regulated sensitivity of Rad53 to activation. This phosphorylation occurs in
G2/M, persists until S phase onset and depends on both the polo-like kinase, Cdc5,
and the cyclin-dependent kinase, Cdc28. These cell-cycle dependent phosphorylation
events are located in the C-terminal part of Rad53. Serines 774 and 789 were shown
to be phosphorylated by mass spectrometry. Mutation of these sites eliminated the
cell-cycle dependent phosphorylation of Rad53 and partially impaired the activation
of Rad53 in response to minor amounts of DNA damage in G2/M. This led to more
rapid checkpoint adaptation in response to irreparable DNA damage. Thus, cell-cycle
dependent phosphorylation in the C-terminal part of Rad53 enhances Rad53
activation in response to DNA double strand breaks (DSBs).
Mec1 and Tel1 initiate a response to DNA damage independently of Rad53. The
phosphorylation of histone H2A at serine 129 (γH2A) at DSBs by Mec1 and Tel1 has
an important role in mediating DNA repair. This study shows that the occurrence of
γH2A is not limited to DSBs, but also occurs at stalled replication forks (Chapter 3).
Using chromatin immunoprecipitation high γH2A levels were monitored at
hydroxyurea-stalled replication forks and depended nearly exclusively upon Mec1
kinase activity. Furthermore our study showed that γH2A not only occurs at damaged
chromatin but in regions of normally replicating chromatin and near telomeres
(Chapter 4). High levels of γH2A could be monitored both in the rDNA of normally
growing yeast cells and at telomeres. Here γH2A depended mainly on Tel1 and
γH2A levels increased during S phase and during the elongation of critically short
telomeres. We also provide evidence that γH2A contributes to telomeric anchoring in
S phase yeast cells in addition to the yKu and Sir4 anchoring pathways.
Advisors:Gasser, Susan
Committee Members:Schär, Primo-Leo
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
UniBasel Contributors:Gasser, Susan and Schär, Primo Leo
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8028
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:166
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:30
Deposited On:29 May 2009 09:07

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