Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Cell Cycle Regulation

Background:

• DNA interstrand crosslinks (ICLs) pose significant threats to cell viability.
• Existing models suggest replication fork convergence initiates ICL repair.
• The precise molecular events following fork convergence remain incompletely elucidated.

Purpose of the Study:

• To investigate the role of replication fork dynamics during DNA interstrand crosslink repair.
• To elucidate the mechanism of ICL unhooking following replication fork convergence.
• To present a revised model for ICL repair incorporating novel intermediates.

Main Methods:

• Utilized Xenopus egg extracts for cell-free DNA interstrand crosslink repair assays.
• Investigated the impact of CMG unloading inhibition on fork progression.
• Analyzed the requirement of FANCI-FANCD2 in fork reversal and DNA incision.

Main Results:

• Observed replication fork reversal at cisplatin-induced ICLs in converging forks.
• Demonstrated that fork reversal is dependent on CMG unloading and replisome disassembly.
• Showed that fork reversal does not require FANCI-FANCD2, but subsequent incisions do.

Conclusions:

• Replication fork reversal is a critical, previously unrecognized step in ICL repair.
• Replisome disassembly is essential for enabling fork reversal at ICLs.
• A revised model incorporating reversed fork intermediates provides a more comprehensive understanding of ICL repair.