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Published July 26, 2019 | Published + Supplemental Material + Submitted
Journal Article Open

RAD51 and mitotic function of MUS81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease


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.

Additional Information

© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 29 August 2018; Revision Received: 02 May 2019; Accepted: 07 May 2019; Published: 22 May 2019. We are grateful to Prof. Massimo Lopes (IMCR, University of Zurich) for scientific discussion. We thank all members of our laboratories for discussion. Authors contributions: F.A.A. performed the analysis of CHK1 phosphorylation, fork recruitment by PLA and chromatin fractionation, and performed experiments to determine DNA damage. A.P. performed the analysis of MUS81 phosphorylation and experiments to evaluate mitotic abnormalities. E.M. analyzed the persistence of RAD51 and parental ssDNA, contributed to the analysis of nascent ssDNA and performed SIRF assays. F.A.A., A.P., E.M. analyzed data, contributed to designing the experiments and writing the manuscript. A.F. and P.P. designed experiments, analyzed data and wrote the paper. L.Z., J.L.C. and B.H.S. provided the DNA2 inhibitor C5, advised the relevant experiments, and revised the manuscript. All authors approved the paper. Funding: Associazione Italiana per la Ricerca sul Cancro (AIRC) [IG17383 to P.P., IG119971 to A.F.]; NIH [R01CA085344 to B.H.S., R50CA211397 to L.Z. and GM123554 to J.L.C.] (in part). Funding for open access charge: AIRC [IG17383 to P.P.]. Conflict of interest statement. None declared.

Attached Files

Submitted - 399881.full.pdf

Published - gkz431.pdf

Supplemental Material - gkz431_supplemental_files.pdf


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August 19, 2023
September 7, 2023