Ski2-like helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

The activating signal co-integrator 1 complex subunit 3 (ASCC3) is a multifunctional protein. However, little is known about its role in replication stress. Here, we report that ASCC3 is recruited to stalled forks by its binding partner ASCC2, whose recruitment to stalled forks requires both its ubiquitin binding activity and polyubiquitylation of PCNA at K164 catalyzed by SHPRH, HLTF, and RFWD3. Upon replication stress, ASCC3 unwinds DNA, and this unwinding activity is required for SMARCAL1 recruitment, restrained fork progression, and fork degradation in BRCA1/BRCA2-deficient cells. ASCC3 unwinds DNA to remodel gap-containing fork substrates in vitro, suggesting that ASCC3 promotes fork reversal. In addition, ASCC3 stimulates RPA accumulation on ssDNA upon replication stress, promoting efficient ATR activation. Furthermore, ASCC3 antagonizes RAD51-mediated recombination and prevents the accumulation of chromosome breaks/gaps and mis-segregation upon replication stress. Our work underscores a critical role of ASCC3 in controlling multiple replication stress responses to maintain genomic stability.