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.

Fig. 1.. Fork reversal does not require CMG disassembly.

(A) iPOND-SILAC mass spectrometry measured abundance changes of selected proteins or complexes comparing HU vs. untreated cells (generated from original data in (14)). nd, not detected. (B) MCM2-AID2 HCT116 cells were labeled with CldU and IdU and treated with 4mM HU for 0-4 hours. Where indicated, 2 μM 5-ph-IAA was added to degrade MCM2 during the HU treatment. Restart efficiency was calculated as the percentage of continuous red and green fibers compared to the total imaged by DNA combing. Mean and SD of three experiments is shown. (C and D) Fork protection assays were completed as indicated. U2OS cells were treated with the inhibitors during the HU treatment time. All graphs are representative of at least three experiments. siNT, non-targeting siRNA. P values were calculated using a Kruskal-Wallis test. (E) iPOND-SILAC-mass spectrometry was used to measure the abundance of proteins at stalled replication forks in HU-treated wild type (WT) and SMARCAL1, ZRANB3, and HLTF triple knockout (3KO) cells. (F) PLA assay for EdU and MCM7 in wild-type or SMARCAL1Δ U2OS cells. (G) PLA of EdU and MCM7 in cells transfected with RAD51 siRNA.

Fig. 2.. RAD51 recombinase activity promotes fork reversal.

(A-C) Fork protection assays were completed in U2OS cells expressing near endogenous levels of the indicated RAD51 proteins after transfection with the indicated siRNAs. P values were calculated using a Kruskal-Wallis test. (EV, empty vector) (D-E) Replication elongation rate in the presence of 50nM camptothecin (CPT) in D) or 150mM cisplatin in E). P values were calculated using a Kruskal-Wallis test. (F) Percentage of reversed replication forks in U2OS cells expressing endogenous levels of the indicated RAD51 proteins. Cells were transfected with the indicated siRNA and treated 72 hours later with 4mM HU, 20μM MRE11 inhibitor (Mirin) and 25μM DNA2 inhibitor (C5) for 5 hours. The number of replication intermediates analyzed for each condition is indicated in parentheses and a representative image is shown (right panel), P: parental strand, D: daughter strand and R: reversed arm.

Fig. 3.. RAD51 is not required for fork reversal when CMG is disassembled from the stalled replication fork.

(A-B) Fork protection assays were completed in MCM2-AID2 degron cells after transfection with siRNAs. 2μM 5-ph-IAA was added to induce MCM2 degradation. (C-D) Immunoblots of MCM3-AID2 and MCM4-AID2 degron cells. (E-F) Fork protection assays in the MCM3-AID2 and MCM4-AID2 degron cells. (G) Immunoblot of GINS4 degron cells. (H) Fork protection assay in the GINS4-AID2 degron cells. (I) MCM7 integrated intensity in the nucleus of GINS4-AID2 degron cells was measured by immunofluorescence. All graphs are representative of at least three experiments. P values were calculated using a Kruskal-Wallis test. (J) Percentage of reversed replication forks in MCM2-AID2 cells transfected with the indicated siRNA and treated 72 hours later with DMSO or 2μM 5-ph-IAA together with 4mM HU, Mirin, and C5 for 5 hours. The number of replication intermediates analyzed for each condition is indicated in parentheses.