Nucleostemin promotes RAD51 filament assembly on double-stranded DNA to protect stalled replication forks.

Replication stress threatens genome integrity by stalling replication forks, which may lead to double-stranded DNA (dsDNA) breaks. Stalled replication forks can be rescued through the fork reversal mechanism, yet they remain vulnerable to nucleolytic attack. Here, we identify nucleostemin (NS/GNL3) as a critical fork-stabilizing factor. We show that NS rapidly accumulates at hydroxyurea-stalled forks and is indispensable for protecting reversed forks, with NS depletion abolishing RAD51 foci formation and unleashing MRE11-mediated degradation. Through biochemical reconstitution and single-molecule fluorescence resonance energy transfer (FRET), we demonstrate that NS binds DNA directly, engages RAD51, and promotes nucleoprotein filament assembly on the dsDNA segment adjacent to the single-stranded DNA/dsDNA junction. This NS-RAD51 complex synergistically shields nascent strands from MRE11-mediated nucleolytic attack. Our work uncovers a new mechanism of fork protection and positions NS as a key guardian of genome integrity under replication stress.