FBH1 Reverses Stalled Replication Forks via Sequential Unwinding of Nascent Strands

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Replication fork reversal is a key DNA damage tolerance mechanism essential for maintaining genome stability.
• F-box helicase 1 (FBH1), a helicase and E3 ubiquitin ligase, is known to catalyze fork reversal and prevent aberrant recombination.
• The precise mechanism by which FBH1's helicase activity drives fork reversal, particularly its role in strand annealing, remained unclear.

Purpose of the Study:

• To elucidate the mechanism by which FBH1 catalyzes DNA replication fork reversal.
• To investigate the role of FBH1's helicase activity in the strand annealing process during fork reversal.
• To determine if FBH1-mediated fork reversal generates a four-way junction and compare it to other known fork reversal proteins.

Main Methods:

• Single-molecule assays were employed to observe DNA replication fork dynamics in real-time.
• Biochemical assays were utilized to analyze the enzymatic activity of FBH1.
• Comparative analysis with other fork reversal proteins like SMARCAL1, HLTF, and ZRANB3 was performed.

Main Results:

• SCF^FBH1 mediates fork reversal via a two-stage mechanism involving translocation on the lagging strand template.
• FBH1 destabilizes the leading strand duplex, displacing the nascent leading strand in a force-sensitive manner.
• This process is annealing-independent and does not generate a four-way junction, distinguishing it from other known mechanisms.

Conclusions:

• FBH1 utilizes nascent strand unwinding as a critical step in replication fork reversal.
• Distinct mechanisms of fork reversal exist, producing unique DNA structures with implications for fork restart and cellular measurement.
• The findings highlight the importance of FBH1 in genome stability and DNA damage tolerance pathways.