Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Replication stress triggers fork reversal, creating DNA ends vulnerable to degradation.
• Fork protection factors (FPFs) prevent this degradation, but their mechanisms are not fully understood.

Purpose of the Study:

• To investigate the role of 53BP1 in protecting reversed replication forks.
• To elucidate the pathways and factors involved in fork protection.

Main Methods:

• Cell-based assays to study replication fork dynamics.
• Genetic manipulation to assess the function of various proteins (53BP1, BRCA2, RAD51, FBH1).
• DNA damage assessment during S-phase.

Main Results:

• 53BP1 protects replication forks from DNA2-mediated degradation in a cell-type-specific manner.
• 53BP1-mediated fork protection reduces S-phase DNA damage and replication stress sensitivity.
• 53BP1 functions via the FBH1 remodeling pathway, distinct from SMARCAL1/ZRANB3/HLTF pathways.
• RAD51 is essential for generating resection substrates in all pathways.
• BRCA2 plays an unexpected role in fork degradation within the FBH1 pathway.

Conclusions:

• Multiple distinct fork protection mechanisms exist, operating downstream of at least two RAD51-dependent fork remodeling pathways.
• 53BP1 represents a novel fork protection factor operating through the FBH1 pathway.