FBH1 Reverses Stalled Replication Forks via Sequential Unwinding of Nascent Strands

Replication fork reversal is a DNA damage tolerance mechanism important for genome stability that entails annealing of parental DNA to push the fork backwards. F-box helicase 1 (FBH1) is a 3'-5' ssDNA translocase and SCF (SKP-CUL1-F-box) E3 ubiquitin ligase that catalyzes fork reversal and limits aberrant recombination, yet how its helicase activity drives strand annealing is unknown. Here, using single-molecule and biochemical assays, we show that SCFFBH1 reverses forks through a two-stage reaction in which translocation on the lagging strand template while remaining affixed at the junction destabilizes the leading strand duplex to ultimately displace the nascent leading strand. Reversal is force-sensitive and does not generate a four-way junction, revealing an annealing-independent mechanism distinct from those of SMARCAL1, HLTF, and ZRANB3. These results establish the importance of nascent strand unwinding to fork reversal and suggest the existence of distinct pathways that produce unique DNA structures, which has implications for fork restart and its measurement in cells.