Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

Abstract

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.

FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability

“…Thus, NA is a functional modulator of FAN1 activity on CAG slip-outs. The endo-and exo-nuclease activities of FAN1 may have distinct functions, as shown for MRE11 and EXO1 (Morafraile et al, 2020;Shibata et al, 2014). We show that blocking one but not other FAN1 functions from acting on repeats can lead to distinct effects, rather than complete ablation of FAN1.…”

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway

“…These results are consistent with the fact that Exo1 is retained at laser-generated DSBs longer in MLH1-deficient cells than in WT cells (Figure 4B), suggesting that higher levels of Exo1 on DNA results in excessive DNA end resection (Figure 3H) and more DSBs (Figure 1C). The current literature reports contradictory roles for pExo1 in the DNA-damage response, with two studies showing that Exo1 phosphorylation activates its activity (Doerfler and Schmidt, 2014;Tomimatsu et al, 2014), one showing Exo1 inactivation (Morafraile et al, 2020), and another showing that pExo1 results in protein degradation (El-Shemerly et al, 2005). Our data suggest that chromatin-bound pExo1 is active in DNA end resection.…”

Hydroxyurea—The Good, the Bad and the Ugly

“…HU activity also leads to the hyper-phosphorylation of the exonuclease 1 (Exo1) needed for resection of stalled or reversed forks that have been irreversibly altered [ 24 , 120 , 121 ]. In humans, Exo1 is phosphorylated in an ATR-dependent manner as well [ 122 , 123 ]. This endonuclease is essential in the process of tempering the 5′ flaps that tend to generate at uncoupled forks and in preventing the generation of reversed forks.…”