RAD51 bypasses the CMG helicase to promote replication fork reversal

Abstract

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

“…In contrast, fork resection was only detected after Aphidicolin treatment in RNH2B‐depleted cells (Fig 7A), which supports the view that RNase H2 is dispensable for the resection of nascent DNA when the replisome is fully blocked. To confirm that this differential effect of HU and Aphidicolin on fork resection was specific to RNH2B‐deficient cells, we next monitored fork resection in the absence of SMARCAL1, a DNA translocase promoting fork reversal (Quinet et al , 2017; Liu et al , 2023) and required for the resection of nascent DNA independently of RNA:DNA hybrids. We found that SMARCAL1‐depleted cells were equally defective for fork resection in the presence of HU or aphidicolin (Fig EV5B) and that the effect of RNaseH2 and SMARCAL1 depletion on resection is epistatic (Fig EV5C).…”

“…It is now well established that stressed forks are extensively remodeled through a process involving fork reversal, in which nascent DNA strands reanneal to form a four‐way structure resembling a Holliday junction (Neelsen & Lopes, 2015). Fork reversal is mediated by the RAD51 recombinase and by different DNA translocases, including SMARCAL1, HLTF and ZRANB3 (Quinet et al , 2017; Liu et al , 2023). Since the regressed arm resembles a one‐ended DSB, it is susceptible to nucleolytic degradation.…”

Disparate requirements for RAD54L in replication fork reversal

“…In response to replication stress, replication forks reverse into four-way junctions through annealing of the nascent DNA strands (2,9). Reversed forks must be protected from nucleolytic attack to prevent fork attrition (10,13,15,48,49).…”

“…The fork reversal activities of SMARCAL1, ZRANB3, and HTLF lead to the degradation of nascent strand DNA in BRCA1/2-deficient cells upon treatment with HU (9,10). Given our results that suggest that RAD54L may function similarly to these established fork remodelers, we tested if RAD54L loss prevents nascent strand DNA degradation in BRCA1/2-deficient cells (Fig.…”

“…Fork reversal creates a 4-way DNA structure and a double-stranded DNA end, and is associated with a slowdown in fork progression, providing time for lesion bypass and repair (3,6). Efficient fork reversal requires the RAD51 recombinase (2,4,7–9), the key protein in homologous recombination DNA repair (HR), and several ATP-dependent DNA motor proteins of the SWI2/SNF2 family including SMARCAL1, ZRANB3, HLTF, and the F-box DNA Helicase 1 (FBH1) (10–14). Stringent regulation of fork reversal is critical for genome stability and dependent on a growing list of fork reversal and protection factors (2,14).…”