Mechanism of replication fork reversal and protection by human RAD51 and RAD51 paralogs

TLDR: It is shown that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal, and the mechanisms of the non-canonical functions of RAD 51 and paralogs in replication fork reversal and protection are defined.

Abstract: RAD51 functions in DNA double-strand break repair by homologous recombination, and by a yet undefined mechanism in the metabolism of challenged replication forks. Here we show that RAD51 directly and specifically promotes the strand annealing and branch migration activities of SMARCAL1 and ZRANB3 but not HLTF, stimulating thus fork reversal. We also find that the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), additionally stimulates SMARCAL1 and ZRANB3 in fork remodeling. DNA binding by RAD51 is required, and the interplay of RAD51, paralogs and the fork remodelers involves direct physical interactions. Upon reversal, RAD51 protects replication forks from degradation by MRE11, DNA2 and EXO1 nucleases. We show that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal. Together, we define the mechanisms of the non-canonical functions of RAD51 and paralogs in replication fork reversal and protection.