Fig 2: HELQ is required for DSB end resection. (A, B) RPA2 phosphorylation was detected by western blotting after the indicated cells were treated with 2 μM camptothecin (CPT, A) or 10 μM etoposide (ETO, B) for the indicated times. (C) The cells were incubated with BrdU for 24 h, then treated with 2 μM CPT for the indicated times, fixed and then stained with BrdU and γH2AX antibodies under native conditions. Representative images (left) and the quantification of the average number of BrdU foci per γH2AX positive cell (right) are shown. scale bar = 10 μm. (D) Indicated cells were treated with CPT (2 μM, 1 h) followed by RPA2 foci formation analysis by immunostaining with the indicated antibodies. Representative images (top) and the quantification of the average number of RPA2 foci per cell (bottom) are shown. Scale bar = 5 μm. (E) Quantification and schematic of qPCR-based end resection assay in HELQ KO cells (#2 and #3). The data represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001. Mann–Whitney test was used in C and D; Student' s t-test was used in E.

Fig 3: HELQ promotes the nuclease activity of EXO1 in vitro. A-B. Nicked plasmid (7 nM) was incubated with purified wild type HELQ (A) or a helicase-dead HELQ mutant (K365M, (B)) in the presence or absence of EXO1 (15 nM) for the indicated times and the products were resolved on a 0.8% agarose gel. (C) 3′-overhang DNA substrate was incubated with the indicated concentration of HELQ and analyzed in 6% native polyacrylamide gel in the presence or absence of EXO1 (20 nM). Protein-DNA complexes are indicated. (D) A structural model of human HELQ-DNA, constructed by superimposing the structure of Archaeoglobus fulgidus HELQ-DNA with human HELQ using AlphaFold2. The enlarged area shows the interaction between human HELQ K587 and ssDNA. (E) Electrophoretic mobility shift assays such as (C) using HELQ variants and ssDNA substrate. The quantification of DNA binding capacity is shown in the bottom. (F) ITC titration and fitting curves of HELQ variants with ssDNA. (G) The effect of HELQ K587A on EXO1 nuclease as described in (A) and (B). In panels A, B, E and G, error bars represent means ± SD of three independent experiments. **P < 0.01, ****P < 0.0001, n.s.: not significant. Student's t-test.

Fig 4: A model illustrating the roles of HELQ in DSB end resection and fork protection. (A) HELQ promotes EXO1-mediated DSB end resection. In the presence of HELQ, EXO1 drives extensive end resection, which is necessary for HR. Without HELQ, EXO1 activity is not fully stimulated, and the ssDNA generated by end resection is insufficient to initiate HR. Both HELQ helicase activity and ssDNA binding ability are needed to promote EXO1 activity in cells. (B) HELQ prevents degradation of nascent DNA on reversed replication forks. HELQ stabilizes RAD51 at reversed forks and limits fork resection by nucleases. Loss of HELQ or its ssDNA binding capacity leads to fork degradation and genomic instability.

Fig 5: HELQ is recruited to DSBs at the early stages of DSB repair. (A) Time-lapse imaging of EGFP-HELQ and EGFP-Ku70 in U2OS cells before and after microirradiation. The red line marks the damage region. (B) Time-lapse imaging of EGFP-HELQ in U2OS cells expressing vector control (Ctrl) or indicated shRNAs before and after microirradiation. C-E. EGFP-MRE11 (C), EGFP- NBS1 (D) and EGFP-CtIP (E) recruitment was monitored in U2OS (WT) and HELQ KO #1 cells. (F) Top, schematic of ChIP assay in ER-AsiSI U2OS cells. Bottom, SFB-HELQ enrichment at DSBs induced by AsiSI enzyme. ER-AsiSI U2OS cells expressing vector control (Ctrl) or the indicated shRNAs were treated with or without 4-OHT (300 nM, 4 h) to induce DSBs before a ChIP assay was performed using an anti-FLAG antibody. In panel A to E, scale bar = 5 μm. In panel F, the data are derived from three independent experiments and represent the means ± SD. *P < 0.05; n.s.: not significant. Student' s t-test was used.