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
Abstract
Abstract 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 characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts 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 BRCA2 in the FA/BRCA fork protection pathway.
Copyright and License
© The Author(s) 2023. 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 (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Acknowledgement
We dedicate this work to the memory of Piotr Polaczek.
Funding
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 RF-2018R1A6A1A0302514 for W.C. sabbatical funding; R50CA211397 to L.Z.; R011CA085344 to B.S.; and USPS grant GM123554 to J.L.C.
Contributions
Author contributions: Conceptualization, J.C.; Methodology, W.L., P.P, I.R, 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 wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
Conflict of Interest
J.C. holds equity in DNATWO, Inc.
Data Availability
The data underlying this article will be shared on reasonable request to the corresponding author.
Supplementary Data are available at NAR Online.
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Additional details
- ISSN
- 1362-4962
- PMCID
- PMC10516637
- National Research Foundation of Korea
- NRF-2017R1A2B2002289
- National Research Foundation of Korea
- NRF-2018R1A6A1A0302514
- National Institutes of Health
- GM123554
- National Institutes of Health
- R50CA211397
- National Institutes of Health
- R011CA085344