Abstract
Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.
Highlights
The response to perturbed replication is crucial for the maintenance of genome integrity [1,2,3,4,5]
The excessive nascent single-stranded DNA (ssDNA) formation detected at 4 h of CPT was recapitulated in doxycycline-inducible shWRN cells complemented with the RNAi-resistant WRNE84A or the WRNK577M-E84A mutant, which contains mutations disabling both the helicase and the exonuclease activities (Supplementary Figure S1A, B)
An increasing interest arose around alternative mechanisms of fork processing and fork degradation since they correlate to the chemosensitivity of cells that are deficient for the primary pathway represented by BRCA1/2-RAD51 [16,18]
Summary
The response to perturbed replication is crucial for the maintenance of genome integrity [1,2,3,4,5]. Proper handling of perturbed replication forks is linked to cancer avoidance and many proteins involved in this process act as onco-suppressors [3,4,5,6]. The importance of correctly dealing with perturbed replication forks is demonstrated by the existence of several human genetic diseases caused by mutations in factors that sense, process and recover replication forks [7]. We recently reported that the exonuclease activity of WRN is involved in protecting replication forks perturbed by treatment with the Topoisomerase I poison Camptothecin (CPT) in the nanomolar range of concentration [12]. BRCA2 and RAD51, are the most crucial for the stabilisation of reversed forks [15,17,18]. BRCA2 and RAD51 may participate in DNA repair,
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