The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair

Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1’s resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks. [Display omitted] •Genome-wide cisplatin sensitivity screens show that SCAI promotes ICL repair•SCAI forms Protexin complexes with REV3, preventing ssDNA accumulation after damage•Protexin loss leads to excessive EXO1 resection of FANCM-reversed forks at ICLs•RNA polymerase interacts with Protexin to restore forks and promote repair Adeyemi et al. performed genome-wide cisplatin sensitivity screens and identified SCAI, which they show to be important for maintaining genome stability following replication stress. They show that SCAI is in complex with a polymerase, REV3, which they term Protexin. Protexin maintains DNA integrity after damage by protecting replication forks from nucleases.