Synergistic protection of nascent DNA at stalled forks by MSANTD4 and BRCA1/2-RAD51

The regressed arms of reversed replication forks exhibit structural similarities to one-ended double-stranded breaks and need to be protected against uncontrolled nucleolytic degradation. Here, we identify MSANTD4 (Myb/SANT-like DNA-binding domain-containing protein 4), a functionally uncharacterized protein that uniquely counters the replication protein A (RPA)-Bloom (BLM)/Werner syndrome helicase (WRN)-DNA replication helicase/nuclease 2 (DNA2) complex to safeguard reversed replication forks from detrimental degradation, independently of the breast cancer susceptibility proteins (BRCA1/2)-DNA repair protein RAD51 pathway. MSANTD4 specifically interacts with the junctions between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in DNA substrates harboring a 3' overhang, which resemble the structural features of regressed arms processed by WRN-DNA2. This DNA-binding capability allows MSANTD4 to accumulate at reversed forks, strategically antagonizing the RPA-BLM/WRN-DNA2 complex by impeding its access to the ssDNA-dsDNA junction of the regressed arms. Loss of MSANTD4 exacerbates genome instability induced by replication stress in BRCA1/2-deficient cells. Our findings unveil a collaborative defense mechanism orchestrated by MSANTD4 and BRCA1/2-RAD51, effectively counteracting nucleolytic attacks on the regressed arms and synergistically preserving the integrity of reversed forks.