Structural basis for fork reversal and RAD51 regulation by the SCF ubiquitin ligase complex of F-box helicase 1

Greer, B. H.; Mendia-Garcia, J.; Mullins, E. A.; Peacock, E. M.; Haigh, S. K.; Aicart-Ramos, C.; Schiltz, C.; Tsai, M.-S.; Cortez, D.; Moreno Herrero, F.; Eichman, B.

doi:10.1101/2025.09.30.679681

bioRxiv · 10.1101/2025.09.30.679681

Structural basis for fork reversal and RAD51 regulation by the SCF ubiquitin ligase complex of F-box helicase 1

Greer, B. H.·Mendia-Garcia, J.·Mullins, E. A.·Peacock, E. M.·Haigh, S. K.·Aicart-Ramos, C.·Schiltz, C.·Tsai, M.-S.·Cortez, D.·Moreno Herrero, F.·Eichman, B.

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Abstract

Replication fork reversal helps maintain genomic stability during replication stress. F-box helicase 1 (FBH1) catalyzes fork reversal and is an SCF (SKP-CUL1-F-box) E3 ubiquitin ligase that limits RAD51 association with chromatin. Here, we show that preferential binding of SCFFBH1 to the lagging strand template at DNA fork structures stimulates helicase activity and is required for fork reversal. A cryo-EM structure of SCFFBH1 bound to DNA representing a stalled fork reveals an intimate interaction between FBH1 and the fork junction. Disruption of this interface severely curtails fork reversal in vitro and replication progression in cells, providing a model for how ssDNA translocation by FBH1 facilitates annealing of parental DNA by a fundamentally different mechanism than the fork remodelers SMARCAL, HLTF, and ZRANB3. The structure also provides a model for SCFFBH1 disassembly of RAD51 filaments through translocation and ubiquitination, and implies that RAD51 is associated with the lagging strand at stalled forks.

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Greer, B. H., Mendia-Garcia, J., Mullins, E. A., Peacock, E. M., Haigh, S. K., Aicart-Ramos, C., Schiltz, C., Tsai, M.-S., Cortez, D., Moreno Herrero, F., Eichman, B.. 2025-10-01. Structural basis for fork reversal and RAD51 regulation by the SCF ubiquitin ligase complex of F-box helicase 1. https://doi.org/10.1101/2025.09.30.679681

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