RADX controls RAD51 filament dynamics to regulate replication fork stability

RADX prevents genome instability by confining replication fork reversal to stalled forks

“…We next examined fork protection in RADXD cells complemented with wild-type, QVPK, or OB2m RADX. As we previously reported, expression of the wild-type RADX protein decreases after a few passages to a level that no longer causes fork deprotection and permits nascent strand degradation when BRCA2 is silenced (Figure 6B; Adolph et al, 2021). In contrast, cells expressing only the RADX QVPK or RADX OB2m proteins have stable forks when BRCA2 is silenced, similar to RADXD cells, consistent with the idea that RADX binding to RAD51 and ssDNA is needed to enable fork reversal in cells experiencing persistent stress (Figures 6B and S5B).…”

“…To test if RADX switched from an antagonist of RAD51 to a positive regulator of RAD51, we purified GFP-FLAG-RADX from unstressed and HU-treated 293T cells (Figure S6A). Both RADX proteins inhibited RAD51-mediated strand exchange and D-loop formation as efficiently as MBP-RADX purified from insect cells, whereas the RADX-QVPK protein did not (Figures S6B and S6C;Adolph et al, 2021). Thus, RADX purified from untreated and HU-treated cells have similar biochemical activities.…”

“…RADX binds RAD51 and ssDNA to promote fork reversal in the presence of persistent replication stress In unstressed cells, the ability of RADX to prevent fork instability is dependent on its direct interactions with RAD51 and ssDNA (Adolph et al, 2021;Dungrawala et al, 2017). To test whether the same mechanisms operate in stressed cells, we analyzed the activities of RADX separation of function mutants with diminished ssDNA binding activity (RADX OB2m; Dungrawala et al, 2017) or an inability to interact with RAD51 (RADX QVPK; Adolph et al, 2021). First, we examined if transient overexpression of these proteins caused nascent strand degradation in HU-treated cells, as previously observed for wild-type RADX (Dungrawala et al, 2017).…”

“…In the absence of added replication stress, RADX inactivation causes slow replication elongation and increased DSBs (Dungrawala et al, 2017;Schubert et al, 2017). RADX competes with RAD51 for ssDNA, directly interacts with RAD51, destabilizes the RAD51 nucleofilament, and inhibits RAD51-dependent processes in vitro (Adolph et al, 2021). Inactivating RAD51 rescues the slow replication elongation and elevated DSBs observed in RADX-deficient cells (Dungrawala et al, 2017).…”

Replication Fork Reversal and Protection

“…Apart from ATR, RAD52 also limits SMARCAL1 activity at stalled forks by counteracting its loading ( Malacaria et al, 2019 ). Moreover, the RPA-like single-strand DNA binding protein RADX antagonizes RAD51 filament formation to prevent inappropriate replication fork reversal ( Dungrawala et al, 2017 ; Schubert et al, 2017 ; Zhang et al, 2020 ; Adolph et al, 2021 ).…”