Header

UZH-Logo

Maintenance Infos

Post-translational modifications regulating Exonuclease 1 in response to replication forks stalling and double-strand breaks


Bologna, Serena. Post-translational modifications regulating Exonuclease 1 in response to replication forks stalling and double-strand breaks. 2014, University of Zurich, Faculty of Science.

Abstract

Double-strand breaks (DSBs), which are among the most dangerous DNA lesions, are estimated to occur at a rate of ten per cell per day in primary human or mouse fibroblasts (Lieber, 2010). These naturally occurring DSBs are generated upon collapse of stalled DNA replication forks, replication across nicks, reactive oxygen species of endogenous origin or the untimely action of DNA endonucleases (topoisomerases or RAG and AID) (Lieber, 2010). Failure to repair damaged DNA has a well-established role in the onset of cancer. Despite the intense effort currently put to the identification of proteins and pathways involved in the recognition of the various forms of DNA damage, we still lack a clear understanding at the molecular level of DNA repair mechanisms and their regulation. In particular, the hierarchy and mutual influence of post-translational modifications (PTMs) on recruitment, function and stability of DNA repair proteins at sites of damage represent new challenges in the field. Appreciating the importance of PTMs will not only allow understanding how dysfunctions of these machineries contribute to the development of cancer and to acquired resistance to therapy, but will also provide the necessary knowledge to target key components of DNA repair pathways and their regulators in the treatment of cancer. This study is aimed to answer important unresolved biological questions regarding the molecular mechanism that controls the function of Exonuclease- 1 (EXO1), a common component of machineries processing stalled replication forks, DSBs and DNA base mismatches. Previous work from our laboratory demonstrated that the function of human and yeast EXO1 at DSBs and stalled forks, respectively, is rigorously controlled by specific protein-protein interactions (Eid et al., 2010; Engels et al., 2011). Additional studies from our laboratory showed that, in response to stalled DNA replication, the cellular level of human EXO1 is regulated by phosphorylation-dependent ubiquitylation that channels EXO1 to proteasome- mediated degradation (El-Shemerly et al., 2008; El-Shemerly et al., 2005). In this study we extend these findings and, taking advantage of a combination of molecular biology and biochemical techniques as well as cell biology 5 assays, we provide new mechanistic insights on the regulation of EXO1. Indeed, by taking advantage of an immunofluorescence-based high- throughput screen followed by image and computational analysis of the acquired data, we identified UBC9-dependent pathways as major effectors of EXO1 stability, indicating that the proteasome-mediated degradation of EXO1 occurring in response to stalled DNA replication is sumoylation-dependent. Moreover we found that the UBC9-PIAS1/PIAS4 pathway controls EXO1 protein stability in vivo both in basal and DNA damaged conditions and we were able to reconstituted EXO1 sumoylation in vitro with purified recombinant human or yeast EXO1 as substrates and components of the sumoylation machinery. The de-sumoylating enzyme SENP6 was found to constitutively interact with EXO1 both in vivo and in vitro and depletion of SENP6 promotes EXO1 degradation. We also showed that sumoylation and ubiquitylation occur sequentially on EXO1 since interfering with the former, by UBC9 depletion or chemical inhibition of the E1-SUMO activating enzyme, compromises the latter. In a second moment, we demonstrated that sumoylation is required for EXO1 recruitment to DNA in response to damage since UBC9 depletion decreases the ratio of chromatin-bound to free EXO1 and the localization of EXO1 at sites of damage, thus resulting in decreased EXO1-mediated resection of DNA ends. In vitro studies combined with mass spectrometric analysis allowed identification of lysine residues K655 and K801/802 as major sumoylation sites in EXO1; Chromosomes spreads analysis from cells expressing high levels of wild-type EXO1 showed a high rate of chromosomes breaks upon camptothecin treatment. This was not the case for cells expressing a SUMO-deficient EXO1 mutant, pointing to an important mechanism that cancer cells with up-regulated EXO1 gene expression may put in place to survive.

Abstract

Double-strand breaks (DSBs), which are among the most dangerous DNA lesions, are estimated to occur at a rate of ten per cell per day in primary human or mouse fibroblasts (Lieber, 2010). These naturally occurring DSBs are generated upon collapse of stalled DNA replication forks, replication across nicks, reactive oxygen species of endogenous origin or the untimely action of DNA endonucleases (topoisomerases or RAG and AID) (Lieber, 2010). Failure to repair damaged DNA has a well-established role in the onset of cancer. Despite the intense effort currently put to the identification of proteins and pathways involved in the recognition of the various forms of DNA damage, we still lack a clear understanding at the molecular level of DNA repair mechanisms and their regulation. In particular, the hierarchy and mutual influence of post-translational modifications (PTMs) on recruitment, function and stability of DNA repair proteins at sites of damage represent new challenges in the field. Appreciating the importance of PTMs will not only allow understanding how dysfunctions of these machineries contribute to the development of cancer and to acquired resistance to therapy, but will also provide the necessary knowledge to target key components of DNA repair pathways and their regulators in the treatment of cancer. This study is aimed to answer important unresolved biological questions regarding the molecular mechanism that controls the function of Exonuclease- 1 (EXO1), a common component of machineries processing stalled replication forks, DSBs and DNA base mismatches. Previous work from our laboratory demonstrated that the function of human and yeast EXO1 at DSBs and stalled forks, respectively, is rigorously controlled by specific protein-protein interactions (Eid et al., 2010; Engels et al., 2011). Additional studies from our laboratory showed that, in response to stalled DNA replication, the cellular level of human EXO1 is regulated by phosphorylation-dependent ubiquitylation that channels EXO1 to proteasome- mediated degradation (El-Shemerly et al., 2008; El-Shemerly et al., 2005). In this study we extend these findings and, taking advantage of a combination of molecular biology and biochemical techniques as well as cell biology 5 assays, we provide new mechanistic insights on the regulation of EXO1. Indeed, by taking advantage of an immunofluorescence-based high- throughput screen followed by image and computational analysis of the acquired data, we identified UBC9-dependent pathways as major effectors of EXO1 stability, indicating that the proteasome-mediated degradation of EXO1 occurring in response to stalled DNA replication is sumoylation-dependent. Moreover we found that the UBC9-PIAS1/PIAS4 pathway controls EXO1 protein stability in vivo both in basal and DNA damaged conditions and we were able to reconstituted EXO1 sumoylation in vitro with purified recombinant human or yeast EXO1 as substrates and components of the sumoylation machinery. The de-sumoylating enzyme SENP6 was found to constitutively interact with EXO1 both in vivo and in vitro and depletion of SENP6 promotes EXO1 degradation. We also showed that sumoylation and ubiquitylation occur sequentially on EXO1 since interfering with the former, by UBC9 depletion or chemical inhibition of the E1-SUMO activating enzyme, compromises the latter. In a second moment, we demonstrated that sumoylation is required for EXO1 recruitment to DNA in response to damage since UBC9 depletion decreases the ratio of chromatin-bound to free EXO1 and the localization of EXO1 at sites of damage, thus resulting in decreased EXO1-mediated resection of DNA ends. In vitro studies combined with mass spectrometric analysis allowed identification of lysine residues K655 and K801/802 as major sumoylation sites in EXO1; Chromosomes spreads analysis from cells expressing high levels of wild-type EXO1 showed a high rate of chromosomes breaks upon camptothecin treatment. This was not the case for cells expressing a SUMO-deficient EXO1 mutant, pointing to an important mechanism that cancer cells with up-regulated EXO1 gene expression may put in place to survive.

Statistics

Downloads

185 downloads since deposited on 25 Nov 2014
57 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Dissertation
Referees:Ferrari Stefano, Lopes Massimo, Penengo Lorenza, Vindigni Alessandro
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:2014
Deposited On:25 Nov 2014 14:05
Last Modified:15 Feb 2018 11:03
OA Status:Green

Download