FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

Liu, W.; Roubal, I.; Polaczek, P.; Meng, Y.; Choe, W.-c.; Caron, M. C.; Sedgeman, C. A.; Xi, Y.; Liu, C.; Wu, Q.; Zheng, L.; Masson, J. Y.; Shen, B.; Campbell, J. L.

doi:10.1101/2021.07.08.450798

bioRxiv · 10.1101/2021.07.08.450798

FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

Liu, W.·Roubal, I.·Polaczek, P.·Meng, Y.·Choe, W.-c.·Caron, M. C.·Sedgeman, C. A.·Xi, Y.·Liu, C.·Wu, Q.·Zheng, L.·Masson, J. Y.·Shen, B.·Campbell, J. L.

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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 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

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Liu, W., Roubal, I., Polaczek, P., Meng, Y., Choe, W.-c., Caron, M. C., Sedgeman, C. A., Xi, Y., Liu, C., Wu, Q., Zheng, L., Masson, J. Y., Shen, B., Campbell, J. L.. 2021-07-09. FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange. https://doi.org/10.1101/2021.07.08.450798

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