RT Journal Article
T1 Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins
A1 Morafraile, Esther C.
A1 Bugallo, Alberto
A1 Carreira Rodríguez, Raquel
A1 Fernández, María
A1 Martín Castellanos, Cristina
A1 González Blanco, Miguel
A1 Segurado, Mónica
AB The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants
PB Oxford University Press
SN 0305-1048
YR 2020
FD 2020
LK http://hdl.handle.net/10347/24486
UL http://hdl.handle.net/10347/24486
LA eng
NO Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Pages 3053–3070, https://doi.org/10.1093/nar/gkaa054
NO Spanish Ministry of Economy and Competitiveness (MINECO) [FEDER-BFU2013-45182-P to C.M.C., M.S.]; University of Salamanca [KA6H/463AC01 to M.S.]; MINECO, AEI, Xunta de Galicia and FEDER [RYC-2012-10835, BFU2016-78121-P, ED431F-2016/019, ED431B-2016/016 to M.G.B.]; Junta de Castilla y León (JCyL), Program ‘Escalera de Excelencia’ [FEDER-CLU-2017-03]; JCyL Pre-doctoral Fellowship (to A.B.); MINECO Pre-Doctoral Fellowship (to E.C.M.); Xunta de Galicia Pre-doctoral Fellowship (ED481A-2018/041 to R.C.)
DS Minerva
RD 25 abr 2026