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Functional characterization of 14-3-3 proteins and exonuclease1 at stalled replication forks


Engels, K. Functional characterization of 14-3-3 proteins and exonuclease1 at stalled replication forks. 2010, University of Zurich, Faculty of Science.

Abstract

DNA replication and DNA repair are two tightly linked processes since,
on the one hand, errors occurring during replication in germline cells might
introduce mutations that are handed on to the next generation and, on the other
hand, unrepaired DNA structures might cause replication blocks that threaten
genome integrity. DNA and replication fork integrity are monitored by
checkpoint-mediated phosphorylation events that trigger repair pathways.
Exonuclease 1 (Exo1) processes stalled replication forks in checkpointdefective
yeast cells. While studying Exo1 and its regulation by
phosphorylation and other post-translational modifications, we isolated an
interesting group of novel in vivo interaction partners in yeast and mammalian
cells, namely the 14-3-3 proteins. 14-3-3’s are able to bind phosphorylated
proteins and, in addition to their well-known ability to act as adaptors that
integrate signals from different pathways, they were shown to play an undefined
role under DNA replication stress. The finding that they interact with Exo1 led
us to formulate the hypothesis that the 14-3-3/Exo1 complex might have a
functional role at replication forks and encouraged us to investigate this
possibility.
Using DNA bi-dimensional electrophoresis, we could show that yeast
14-3-3’s promote fork progression under limiting nucleotide concentrations. 14-
3-3-deficient cells fail to induce Mec1-dependent Exo1 hyper-phosphorylation
and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by
electron microscopy. This leads to persistent checkpoint activation and
exacerbated recovery defects. Interestingly the fork progression defect in 14-3-3
cells cannot be rescued by Exo1 deletion and the recovery defect is only
partially rescued by Exo1 deletion, suggesting that additionally to Exo1, 14-3-3
proteins might regulate the phosphorylation of other yet unknown targets in
response to replication fork stalling. Based on this evidence, we propose that 14-3-3 proteins assist
checkpoint-mediated phosphorylation of Exo1 and additional unknown targets,
promoting fork progression, stability and restart in response to DNA replication
stress.

Abstract

DNA replication and DNA repair are two tightly linked processes since,
on the one hand, errors occurring during replication in germline cells might
introduce mutations that are handed on to the next generation and, on the other
hand, unrepaired DNA structures might cause replication blocks that threaten
genome integrity. DNA and replication fork integrity are monitored by
checkpoint-mediated phosphorylation events that trigger repair pathways.
Exonuclease 1 (Exo1) processes stalled replication forks in checkpointdefective
yeast cells. While studying Exo1 and its regulation by
phosphorylation and other post-translational modifications, we isolated an
interesting group of novel in vivo interaction partners in yeast and mammalian
cells, namely the 14-3-3 proteins. 14-3-3’s are able to bind phosphorylated
proteins and, in addition to their well-known ability to act as adaptors that
integrate signals from different pathways, they were shown to play an undefined
role under DNA replication stress. The finding that they interact with Exo1 led
us to formulate the hypothesis that the 14-3-3/Exo1 complex might have a
functional role at replication forks and encouraged us to investigate this
possibility.
Using DNA bi-dimensional electrophoresis, we could show that yeast
14-3-3’s promote fork progression under limiting nucleotide concentrations. 14-
3-3-deficient cells fail to induce Mec1-dependent Exo1 hyper-phosphorylation
and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by
electron microscopy. This leads to persistent checkpoint activation and
exacerbated recovery defects. Interestingly the fork progression defect in 14-3-3
cells cannot be rescued by Exo1 deletion and the recovery defect is only
partially rescued by Exo1 deletion, suggesting that additionally to Exo1, 14-3-3
proteins might regulate the phosphorylation of other yet unknown targets in
response to replication fork stalling. Based on this evidence, we propose that 14-3-3 proteins assist
checkpoint-mediated phosphorylation of Exo1 and additional unknown targets,
promoting fork progression, stability and restart in response to DNA replication
stress.

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Additional indexing

Item Type:Dissertation
Referees:Lopes M, Ferrari S, Paszkowski J
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2010
Deposited On:31 Oct 2011 13:29
Last Modified:26 Jan 2017 08:49
Number of Pages:98
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=006365060

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