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Published July 12, 2021 | Submitted + Supplemental Material
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FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability


FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We showed that FANCD2 inhibits fork degradation in two ways: 1) the N-terminal domain of FANCD2 inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11, and EXO1. Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts as a "RAD51 modulator" to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator in the FA/BRCA pathway of fork protection.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Version 1 - July 9, 2021; Version 2 - August 5, 2022. This work was supported by a CIHR foundation grant (J.-Y.M.) and J.-Y.M. is a FRQS Chair in genome stability; Korean government grants NRF-2017R1A2B2002289 and NRF-2018R1A6A1A0302514 for W.C. sabbatical funding; R50CA211397 to L.Z.; R011CA085344 to B.S.; and USPS grant GM123554 to JLC. Author Contributions: Conceptualization, J.C.; Methodology, W.L., I.R., P.P., Y.M., M.C., Y.X., C.L., Q.W., L.Z.; Writing – Original Draft, W.L., P.P., J.C.; Writing – Review and Editing, W.L.,P.P, L.Z., J.M., B.S, J.C.; Funding Acquisition, W.C., J.M., B.S., J.C; Supervision J.M., B.S., J.C. The authors declare no competing interests.

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Submitted - 2021.07.08.450798v2.full.pdf

Supplemental Material - media-1.pdf


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August 20, 2023
January 17, 2024