Human RAD52 double-ring remodels replication forks restricting fork reversal.

Human RAD52 1,2 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation 3-7 . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1 5 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.

RAD52 prevents accumulation of Polα-dependent replication gaps at perturbed replication forks in human cells.

Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polα downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polα is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polα and RAD51 is stimulated by RAD52 inhibition, and Polα-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polα-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled.