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

[摘要] DNA replication and DNA repair are two tightly linked processes since,on the one hand, errors occurring during replication in germline cells mightintroduce mutations that are handed on to the next generation and, on the otherhand, unrepaired DNA structures might cause replication blocks that threatengenome integrity. DNA and replication fork integrity are monitored bycheckpoint-mediated phosphorylation events that trigger repair pathways.Exonuclease 1 (Exo1) processes stalled replication forks in checkpointdefectiveyeast cells. While studying Exo1 and its regulation byphosphorylation and other post-translational modifications, we isolated aninteresting group of novel in vivo interaction partners in yeast and mammaliancells, namely the 14-3-3 proteins. 14-3-3’s are able to bind phosphorylatedproteins and, in addition to their well-known ability to act as adaptors thatintegrate signals from different pathways, they were shown to play an undefinedrole under DNA replication stress. The finding that they interact with Exo1 ledus to formulate the hypothesis that the 14-3-3/Exo1 complex might have afunctional role at replication forks and encouraged us to investigate thispossibility.Using DNA bi-dimensional electrophoresis, we could show that yeast14-3-3’s promote fork progression under limiting nucleotide concentrations. 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyper-phosphorylationand accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed byelectron microscopy. This leads to persistent checkpoint activation andexacerbated recovery defects. Interestingly the fork progression defect in 14-3-3cells cannot be rescued by Exo1 deletion and the recovery defect is onlypartially rescued by Exo1 deletion, suggesting that additionally to Exo1, 14-3-3proteins might regulate the phosphorylation of other yet unknown targets inresponse to replication fork stalling. Based on this evidence, we propose that 14-3-3 proteins assistcheckpoint-mediated phosphorylation of Exo1 and additional unknown targets,promoting fork progression, stability and restart in response to DNA replicationstress.

[发布机构] University of Zurich

[效力级别] 570 Life sciences

[关键词] Institute of Molecular Cancer Research;570 Life sciences;biology