HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

To investigate the functions of HELQ, we purified recombinant human HELQ from insect cells (Extended Data Fig. 1a), which efficiently unwound substrates containing 3′ overhangs or a D-loop (Fig. 1a, b and Extended Data Fig. 1b-d). As previously reported, HELQ prefers to unwind single and double-stranded DNA junctions and therefore showed greater unwinding of 3′ overhangs and Y-structures than 3′ lagging strand forks and D-loops 7 . However, at higher concentrations of HELQ, no unwound product was observed, especially for substrates containing 3′ overhangs (described below; Extended Data Fig. 1e). HELQ showed no unwinding with ATPγS, a poorly hydrolysable ATP analogue, and failed to unwind dsDNA and 5′ overhang substrates (Extended Data Fig. 1f-h). The helicase-dead mutant of HELQ (HELQ(K365M)) lacked DNA-unwinding activity and showed slightly increased binding to ssDNA and dsDNA compared with the wild-type (WT) protein (Extended Data Fig. 1a, i-m).

RAD51 stimulates HELQ unwinding activity
In vivo studies have shown that HELQ-deficient cells exhibit persistent RAD51 foci after DNA damage 3,8 . Furthermore, HELQ-1 from Caenorhabditis elegans interacts with RAD-51 (ref. 8 ). HELQ and human RAD51, purified from Escherichia coli (Extended Data Fig. 2a), also interact directly (Extended Data Fig. 2b). In unwinding assays, RAD51 strongly stimulated HELQ helicase activity with all of the tested substrates, whereas bacterial RecA-an orthologue of RAD51-did not stimulate HELQ even at higher concentrations ( Fig. 1c-e and Extended Data Fig. 2c-g). We next purified and tested the BRC4 peptide (single BRCA2 BRC repeat), which prevents RAD51 from binding to DNA (Extended Data Fig. 2h-j). The BRC4 peptide did not inhibit stimulation of HELQ by RAD51(Extended Data Fig. 2k-n), indicating that RAD51 DNA binding is not required for HELQ stimulation and excludes DNA sequestration as a possible mechanism for stimulation by RAD51. We observed that, at a higher concentration of RAD51 (that is, 120 nM), HELQ unwinding activity is inhibited (Fig. 1c, d).
To investigate this, we tested the BRC4 peptide with an excess RAD51 and found that inhibiting RAD51 DNA binding also rescued DNA unwinding by HELQ (Extended Data Fig. 2o, p). Furthermore, by measuring the kinetics of DNA unwinding by HELQ, we found that addition of RAD51 resulted in a concentration-dependent increase in the HELQ DNA unwinding rate, whereas addition of RecA had no effect (Extended Data Fig. 2q-s). In cells, ssDNA generated during DNA processing is bound by RPA. To mimic these conditions, we purified fluorescently tagged human RPA-mRFP1 from E. coli (Extended

Visualization of HELQ DNA unwinding
To better understand HELQ stimulation by RAD51, we used an optical tweezer set-up combined with microfluidics and confocal microscopy (C-TRAP) for single-molecule imaging (SMI) analysis. As shown in Fig 1h, a single dsDNA molecule (λ-DNA) containing a ssDNA gap 9 was tethered between two optically trapped beads and held at constant force (50 pN) to prevent the reannealing of unwound DNA. After addition of HELQ, DNA unwinding was observed as an increase in the distance between the beads due to the expansion of the ssDNA region. Neither RAD51 alone nor HELQ(K365M) showed evidence of unwinding ( Fig. 1i and Extended Data Fig. 4a). Combining WT HELQ and RAD51 resulted in a considerable increase in overall DNA unwinding, whereas no such stimulation was observed with HELQ(K365M) (Fig. 1j, k and Extended Data Fig. 4b). Within unwinding traces for individual DNA molecules, rapid unwinding bursts interspersed by pauses can be distinguished (Extended Data Fig. 4c-e) and corresponded to a mean rate of 3.3 ± 0.4 nm s −1 (mean ± s.e.m Extended Data Fig. 4f).
In the presence of RAD51, an increased number of molecules showed greater unwinding rates (Extended Data Fig. 4g). To directly visualize RAD51 during DNA unwinding with HELQ, mutant RAD51(C319S) was purified and efficiently labelled with Alexa Fluor 488 C5 maleimide dye (Alx-RAD51) (Extended Data Fig. 4h). Whereas Alx-RAD51 alone displayed mostly static binding traces with occasional diffusing species, addition of HELQ showed unidirectionally translocating traces indicating active movement of an Alx-RAD51-HELQ complex along the ssDNA backbone ( Fig. 1l and Extended Data Fig. 4i). After analysis, we found that HELQ with RAD51 translocates fastest at the rate of 14 ± 5 nm s −1 in gapped substrate (Extended Data Fig. 4j, k). By contrast, HELQ(K365M) retained the ability to bind to RAD51 but showed no translocation with only static or diffusing traces. Together, these results indicate that RAD51 and HELQ form a complex that unwinds DNA at a rate of approximately threefold faster compared with HELQ alone.

DNA strand annealing by HELQ
As shown above, a strong reduction in unwound product was observed at higher concentrations of HELQ (Fig. 1a, b and Extended Data Fig. 1b-e); we reasoned that this could result from the reannealing of the unwound product. Indeed, reactions containing an unlabelled 'cold' oligonucleotide (oligo) yielded an increase in unwound product with excess HELQ (Extended Data Fig. 5a (compare lanes 3 and 4 with 7 and 8)). Kinetic analysis also showed that HELQ initially unwinds the substrate but then reanneals it back together at later time points (Extended Data Fig. 5b). Prompted by this, we directly tested HELQ for DNA strand annealing activity without and with an excess of RPA needed for 100% ssDNA coverage (theoretically, 16 nM RPA covers 10 nM ssDNA). We found that HELQ efficiently anneals complementary DNA strands either without or with RPA ( Fig. 2a, b). At lower concentrations of HELQ, RPA stimulated HELQ DNA annealing activity by around twofold. However, at higher concentrations, HELQ showed greater DNA annealing activity in the absence of RPA. This raised the possibility that RPA aids HELQ loading on ssDNA when HELQ is present in limiting amounts. Titration experiments confirmed that substochiometric levels of RPA are sufficient to stimulate HELQ annealing activity (Fig. 2c, d). Notably, HELQ could still anneal complementary DNA strands in the presence of excess RPA (Extended Data Fig. 5c, d). We next tested the requirement of ATP binding and hydrolysis for DNA annealing by HELQ. Surprisingly, in the presence of RPA, HELQ showed no DNA annealing without ATP, whereas ATP became dispensable when RPA was excluded from the reaction (Fig. 2e, f and Extended Data Fig. 5e, f). Even in the absence of RPA, ATP stimulated the DNA annealing activity of HELQ (Extended Data Fig. 5e, f). HELQ also failed completely to anneal DNA with ATPγS in the presence of RPA (Extended Data Fig. 5g). Collectively, these data suggest that HELQ possesses intrinsic DNA annealing activity that requires ATP binding and hydrolysis when ssDNA is coated with RPA. We next tested the helicase-inactive HELQ(K365M) mutant for DNA annealing activity and found that HELQ(K365M) is defective for DNA annealing in the presence of excess RPA but could anneal ssDNAs when RPA is excluded from reactions (Extended Data Fig. 5h, i). A titration experiment showed that, in contrast to the WT, HELQ(K365M) becomes progressively impaired by increasing concentrations of RPA (Fig. 2g, h and Extended Data Fig. 5j, k). RPA also failed to stimulate HELQ(K365M) (Fig. 2g, h). We also tested E. coli SSB protein and found that it only weakly stimulates HELQ annealing activity (Extended Data Fig. 5l, m). The N-terminal fragment of HELQ was previously shown to displace RPA from ssDNA 6 . However, full-length HELQ was not analysed for such activity. Thus, we directly tested RPA displacement from ssDNA during DNA strand annealing by omitting the deproteination step. We observed that HELQ can strip an excess of RPA from ssDNA, which occurred coincidently with the appearance of the annealed products (Extended Data Fig. 6a, b). To directly visualize RPA stripping from ssDNA by HELQ, we measured RPA-eGFP displacement by SMI analysis and found that WT HELQ (k = 0.136 ± 0.008 min −1 ) could efficiently strip RPA from ssDNA but HELQ(K365M) (k = 0.017 ± 0.004 min −1 ) could not ( Fig. 2i-k). Using a single-molecule Förster resonance energy transfer (FRET)-based assay (Extended Data Fig. 6c-j), we observed concentration-dependant RPA stripping by WT HELQ, followed by rebinding of RPA (Extended Data Fig. 6k-n). The RPA rebinding is independent of HELQ concentration, indicating a constant transition rate from the free (t on ) to bound (t off ) state at various HELQ concentrations (Extended Data Fig. 6o). HELQ(K365M) did not show any RPA stripping (Extended Data Fig. 6p), indicating that active RPA stripping has a critical role in HELQ-mediated DNA annealing. Finally, we found that the addition of RAD51 had no effect on HELQ-dependent DNA annealing activity (Extended Data Fig. 6q, r). To study whether, like RPA, HELQ can also strip RAD51 from ssDNA, we directly tested RAD51 removal from ssDNA and dsDNA. HELQ did not remove RAD51 from either ssDNA or dsDNA in bulk assays (Extended Data Fig. 7a-d) and only weakly displaced RAD51 from ssDNA in our SMI setup (Extended Data  . This suggests that HELQ is unlikely to disrupt fully formed RAD51 nucleofilaments but might remove RAD51 that is bound to short-resected DNA, as is present during microhomology-mediated end joining (MMEJ).

HELQ captures RPA-ssDNA
RAD52 has been shown to possess robust DNA strand annealing activity and has a central role in single-strand annealing (SSA) repair of DSBs 10-14 .
To investigate the mechanistic basis of the strand annealing ability of HELQ, we modelled our experiments on RAD52 annealing activity. Using optical tweezers, it was previously shown that RAD52 can trans-capture labelled oligos at multiple sites along -DNA, independent of DNA sequence 15 . Using a similar set-up, we tested the ability of HELQ to capture a Cy3-labelled 80-mer oligo ssDNA in the presence of RPA-eGFP (around 100% coverage; Fig 3a and Extended Data Fig. 8a). HELQ facilitated the capture of 4 oligo 16,17 at multiple sites (Fig. 3b, c). Notably, in contrast to its annealing or unwinding activity, HELQ(K365M) exhibited efficient DNA capture activity (Fig. 3b, c). We also analysed the dwell times of captured oligos and found that HELQ(K365M) showed moderately increased dwell times compared with the WT (Fig. 3d, e), most likely due to slightly higher DNA binding of HELQ(K365M). We also tested a 79-nucleotide T-homopolymer (dT79) and obtained similar capture frequencies as with the 4 oligo (Fig. 3c).
HELQ showed capture of both homologous (λ4) and T-homopolymer oligos at various sites on λ-DNA. To determine whether microhomologies present in ~50 kb λ-DNA could explain the capture pattern of both oligos, we performed forced-stretching experiments as shown in Fig. 3f. As DNA starts to melt at forces of >60 pN, we reasoned that, if HELQ oligo capture involves base-pairing interactions, short microhomologies should dissociate faster than the ones with longer homology. However, even at very high force (90-100 pN), all oligos remained engaged with ssDNA, irrespective of position (Fig. 3g). We next performed pulling experiments 18 and observed characteristic force spikes when beads were pulled apart at a low force (10-15 pN) (Extended Data Fig. 8b, c). These spikes correspond to the disruption of HELQ complexes capturing RPA-coated ssλ-DNA in cis. To examine this further, we developed a bulk capture assay, in which we attempted to pull out labelled non-complementary DNA (Cy3-dT79) with a biotinylated dT43 oligo with HELQ. We found that both WT HELQ and the HELQ(K365M) mutant could capture non-complementary oligos bound to RPA (Fig. 3h, i and Extended Data Fig. 8d). Thus, HELQ and HELQ(K365M) can both capture DNA strands independent of sequence, probably through DNA tethering, but only the WT can anneal RPA-coated complementary DNA strands through RPA stripping. Interestingly, yeast Rad52, when bound to RPA-coated ssDNA clusters, can capture additional free

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RPA in pre-existing Rad52-RPA-ssDNA clusters 19 . This activity was postulated to be important for second-end capture during homologous recombination (HR).

HELQ functions in SSA and MMEJ
To extend our findings with HELQ to DSB repair in vivo, we first confirmed that deletion of HELQ or protein depletion in cells inhibits HR (Extended Data Fig. 9a-e). As DNA annealing is required for SSA repair, we investigated a potential role for HELQ in this process. Strikingly, HELQ depletion or HELQ deletion impaired SSA repair of an integrated SSA reporter (SA-GFP; Fig. 4a, b). Although depletion of the HR factor, BRCA2, increases SSA repair, this was strongly reduced by HELQ depletion ( Fig. 4c and Extended Data Fig. 9f). Consistent with an epistatic role in SSA, co-depletion of RAD52 and HELQ did not further decrease SSA repair compared with individual depletions (Extended Data Fig. 9g). We also assessed whether HELQ functions in alternative end joining repair, which also involves an annealing step. Using cells containing both EJ-RFP and DR-GFP reporter systems for simultaneous detection of alternative end joining and HR, respectively, we observed a significant reduction in both DSB repair pathways after HELQ depletion (Fig.4d, e). Alternative end joining encompasses broad non-nonhomologous end joining (NHEJ) repair events including MMEJ. To specifically study the role of HELQ in MMEJ, we used a Cas9-mediated DSB repair assay in which the fate of DSB repair by MMEJ, NHEJ or single-stranded templated repair (SSTR) can be determined 20 (Fig. 4f). The loss of HELQ resulted in a slight increase in NHEJ, whereas MMEJ was significantly impaired and SSTR was completely abolished (Fig. 4g)  Data are mean ± s.e.m. h, Schematic of the RFP-SCR reporter assay. Tr GFP, truncated GFP. i, I-SceI-induced STGC frequency in U2OS-RFP-SCR cells treated with the indicated siRNA. n = 13 (siNT) n = 11 (siHELQ 1), n = 12 (siRAD52) and n = 6 (siHELQ 1/siRAD52) independent experiments. Data are mean ± s.e.m. j, LTGC frequency as shown in i. n = 13 (siNT), n = 12 (siHELQ 1), n = 11 (siRAD52) and n = 6 (siHELQ 1/siRAD52) independent experiments. Data are mean ± s.e.m. k, The ratio of LTGC/total GC from the experiments in i and j. n = 14 (siNT), n = 12 (siHELQ 1), n = 12 (siRAD52) and n = 6 (siHELQ 1/siRAD52) independent experiments. Data are mean ± s.e.m. For b, c, e, g, i-k, statistical analysis was performed using two-tailed paired t-tests as compared with siNT, unless otherwise indicated. moderate reduction in short-tract gene conversion (STGC) and LTGC, respectively, with the LTGC/total gene conversion (GC) ratio showing a significant bias towards LTGC (Fig. 4h-k). Finally, co-depletion of HELQ with RAD52 showed a further bias towards LTGC, implying that they have roles in parallel pathways during GC (Fig. 4k).
In summary, our study implicates HELQ in several distinct DSB repair pathways, including HR, SSA and MMEJ, casting light on its role in genome stability and tumour avoidance. As these repair pathways each require DNA annealing steps, we propose that HELQ functions in these pathways through its intrinsic ability to capture RPA-bound ssDNA and then displace RPA to facilitate annealing of complementary DNA strands. HELQ alone or in complex with RAD51 might unwind D-loops before annealing complementary strands (Extended Data Fig. 10). The bias towards LTGC events after HELQ depletion is consistent with a role for DNA annealing by HELQ during second-strand capture and/or synthesis-dependent strand annealing during HR, which may explain why HR is reduced in HELQ-deficient cells. Finally, our finding that HELQ is epistatic with RAD52 for SSA but additive for GC during HR is surprising, as this implicates two DNA strand annealing enzymes in several distinct DSB repair processes in cells.

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Preparation of expression vectors
Custom HELQ ORF was purchased from GeneArt and used as a template during PCR to prepare plasmid (pFastbac1) compatible for bacmid preparation for expression in insect cells. To prepare the MBP-HELQ-Flag construct, HELQ was amplified by PCR using the primers HELQ_F and HELQ_FLAG_R. The amplified insert was digested with BamHI and XmaI and inserted into the pFastbac1 vector containing MBP (maltose binding protein tag; previously inserted using the EcoRV and HindIII restrictions sites). The resulting construct was pFB-MBP-HELQ-FLAG. To prepare helicase-dead HELQ(K365M), pFB-MBP-HELQ-Flag was mutagenized with the primers HELQ_K365M_F and HELQ_K365M_R using the Q5 site-directed mutagenesis kit according to the manufacturer's instructions. The constructs for pET11c-RAD51 and, RPA-eGFP (pMM801) and RPA-mRFP1 (pMM802) were gifts from L. Krejci and M. Modesti, respectively.

Recombinant protein purification
To express proteins in insect cells, bacmid, primary and secondary baculoviruses were prepared according to the manufacturer's instructions (Bac-to-bac system, Life technologies). To express recombinant MBP-HELQ-FLAG, Spodoptera frugiperda (Sf9) insect cells were seeded at 500,000 cells per ml and, after around 24 h, cells were infected with MBP-HELQ-Flag baculovirus. The infected cells were incubated at 27 °C for 56 h with continuous agitation. Cells were collected by centrifugation at 500g for 10 min and washed once with 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ). The collected pellets were snap-frozen in liquid nitrogen and stored at −80 °C until further use. All subsequent steps were performed either on ice or at 4 °C. The cells pellets were resuspended in 3 volumes of lysis buffer containing 50 mM Tris-HCl pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail tablets (Roche), 30 µg ml −1 leupeptin (Merck), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 0.1% NP-40 substitute (NP-40) and incubated for 15 min with continuous agitation. Next, 50% glycerol and 5 M NaCl were added sequentially to the final concentrations of 16.7% and 310 mM, respectively, and the suspension was further incubated for 30 min with continuous agitation. The suspension was centrifuged at ~48,000g for 30 min to obtain the soluble extract. The amylose resin (NEB) was pre-equilibrated with amylose wash buffer I (50 mM Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol (β-ME), 1 M NaCl, 1 mM PMSF, 10% glycerol and 0.1% NP-40) and added to 50 ml tubes containing soluble extract. These tubes were subsequently incubated for 1 h with continuous rotation. After incubation, the resin was washed batchwise four times by centrifugation at 2,000g for two min and twice on column with amylose wash buffer I. Resin was washed twice more on column with amylose wash buffer II (same as wash buffer I but with 0.5 mM β-ME and 0.8 M NaCl). Protein was eluted from the resin using amylose elution buffer (same as amylose wash buffer II supplemented with 10 mM maltose) and total protein was estimated using the Bradford assay. To remove the MBP tag, 1/8 (w/w) of PreScission protease to the total protein was added to amylose eluate and incubated for 2 h without rotation but with gentle agitation at regular intervals. The Flag resin (anti-Flag M2 resin, Sigma-Aldrich), which was pre-equilibrated with Flag wash buffer (50 mM Tris-HCl pH 7.5, 0.8 M NaCl, 1 mM PMSF,10% glycerol), was added to amylose eluate containing PreScission protease and incubated for 2 h with continuous rotation. Flag resin was collected directly on column and washed six times with Flag wash buffer. The protein was eluted from Flag resin with Flag elution buffer (50 mM Tris-HCl pH 7.5, 0.5 mM β-ME, 150 mM NaCl, 1 mM PMSF,10% glycerol, 150 ng µl −1 3×Flag peptide (Sigma-Aldrich)), aliquoted, frozen in liquid nitrogen and stored at −80 °C. The same purification procedure was used to purify HELQ(K365M).
Recombinant human RAD51 was purified as described previously with a few modifications 30 . The pET11c-RAD51 expression vector was transformed into E. coli BLR(DE3)pLysS cells and subsequent culture containing ampicillin (100 mg l −1 ) and chloramphenicol (33 mg l −1 ) was grown to an optical density (OD) at 600 nm of 0.7. RAD51 expression was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 3-4 h. All of the subsequent steps were performed either on ice or at 4 °C. Cells were collected by centrifugation at 5,000g. Cell pellets were resuspended in cell breakage buffer (50 mM Tris-HCl pH 7.5, 10% sucrose, 0.5 mM EDTA, 1 M KCl, 1 tablet per 50 ml of protease inhibitor cocktail tablets (Roche), 1 mM PMSF, 1 mM DTT and 0.01% NP-40), sonicated and centrifuged at 100,000g for 1 h. To precipitate RAD51, 0.242 g ml −1 ammonium sulphate was mixed with clarified supernatant and centrifuged for 20 min at 10,000g. The pellet was resuspended with buffer K (20 mM K 2 HPO 4 pH 7.5, 10% glycerol, 0.5 mM EDTA, 1 mM DTT, 0.01% NP-40) and loaded onto the Q Sepharose Fast flow column (Cytiva), pre-equilibrated with K buffer-low (K buffer supplemented with 175 mM KCl). The column was washed extensively with K buffer-low and protein was subsequently eluted with a KCl gradient using K buffer-high (K buffer supplemented with 0.6 M KCl). The eluted fractions containing RAD51 were pooled and diluted with 6 volumes of dilution buffer (25 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 mM DTT, 0.01% NP-40). The diluted sample was loaded onto the HiTrap Heparin HP affinity column (Cytiva), which was pre-equilibrated with buffer H without glycerol (25 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 mM DTT, 0.01% NP-40, 150 mM KCl) and washed with buffer H containing 10% glycerol. Protein was eluted in buffer H with a KCl gradient (0.1 M to 1 M KCl) and the fractions containing RAD51 were pooled and dialysed in buffer H without glycerol. The dialysed sample was loaded onto the Mono Q 5/50 GL column (Cytiva), equilibrated with buffer Q (25 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 mM DTT, 0.01% NP-40, 100 mM KCl, 10% glycerol) and the column was further washed with buffer Q containing 50 mM KCl but lacking glycerol. RAD51 was eluted from the Mono Q column with a KCl gradient (0.05 M to 1 M) in buffer Q lacking glycerol. The eluted fractions containing RAD51 were pooled and further concentrated with the Vivaspin Centrifugal Concentrator (30 kDa molecular weight cut-off (MWCO)). Glycerol was added to the concentrated sample to a final concentration of 10%. Finally, the samples were aliquoted, frozen in liquid nitrogen and stored at −80 °C. The RAD51(C319S) mutant was purified using same procedure. RPA-mRFP1 and RPA-eGFP were purified as described previously 31 . Recombinant RecA (M0249) and ET SSB (M2401) were commercially purchased from NEB, England.
To purify GST and GST-BRC4, the BRC4 construct was cloned into the pGEX6P-1 vector containing a GST tag using BamHI and EcoRI restriction sites. The pGEX6P-1 and pGEX6P-1-BRC4 constructs were transformed into BL21 DE3 cells and plated onto an agar plate containing ampicillin (100 µg ml −1 ). A single colony was isolated and inoculated into 6 ml preculture overnight. The next day, 4.5 ml preculture medium was added to 450 ml LB medium containing ampicillin (100 µg ml −1 ), and the OD at 600nm was monitored at regular intervals. Proteins were induced with 1 mM IPTG at 0.6 OD and cultures were incubated for 4 h. The cell pellets were collected, washed with cold PBS and stored at −80 °C. For purification, the cell pellets were lysed by sonication in PBS and the samples were centrifuged at 75,600g for 30 min at 4 °C. Next, the supernatant was collected and incubated with 1.2 ml glutathione resin for 1 h at 4 °C. The resin was washed three times with PBS and the proteins were eluted with elution buffer (20 mM Tris-HCl pH 7.5, 20 mM glutathione). The eluted proteins were aliquoted, snap-frozen in liquid nitrogen and stored at −80 °C.

Preparation of labelled proteins
The RAD51(C319S) variant was expressed and purified as described earlier for WT RAD51 (refs. 30,32 ). After purification, the protein was fluorescently labelled with Alexa Fluor 488 C 5 maleimide (Thermo Fisher Scientific, A10254) according to previously described protocol 31 .
Labelled protein was purified away from the free dye using the Zeba column gel filtration system (0.5 ml resin, 50 kDa MWCO). The protein concentration was estimated by Coomassie staining and dye concentration was measured spectrophotometrically. The presence of minimum free dye concentration was assessed using SDS-PAGE on labelled proteins. The protein to dye concentration ratio was consistently 0.9-1.0. D-loop formation of labelled RAD51 was tested and gave yields comparable to unlabelled WT RAD51 protein, consistent with previous reports 32 . RPA-eGFP and RPA-mRFP1 were expressed and purified as described previously 31 . DNA binding of labelled RPA was tested. All RPA-fusion proteins displayed similar ssDNA affinities within nanomolar K d range.

DNA unwinding assay
The unwinding assays were performed in 15 µl helicase buffer containing 25 mM Tris-HCl pH 8.0, 2 mM ATP, 2 mM MgCl 2 , 1 mM DTT, 50 mM NaCl, 0.1 mg ml −1 bovine serum albumin (BSA, New England Biolabs), 1 mM PEP (phosphoenolpyruvate, Sigma-Aldrich), 10 U ml −1 pyruvate kinase (Sigma-Aldrich) and 5′-end-FITC-labelled 25 nM DNA substrate (in molecules). All of the steps except the assembling reactions and protein addition were performed in the dark. The reactions were assembled on ice and recombinant proteins were added, mixed and incubated at 37 °C for 30 min. The reactions were stopped with 5 µl of 2% stop solution (0.2% SDS, 30% glycerol, 150 mM EDTA, bromophenol blue) and 1 µl proteinase K (Roche, 18.4 mg ml −1 ) and incubated for 10 min at 37 °C. To prevent reannealing, 2% stop solution was supplemented with tenfold excess of unlabelled oligos with the same sequence as the FITC-labelled oligo. The products were resolved by 10% native polyacrylamide gel (19:1 acrylamide-bisacrylamide, Bio-Rad) using Mini-Protean Tetra Cell electrophoresis system (Bio-Rad) at 100 V for 1 h. The gels were directly imaged in ChemiDoc MP imaging system.

Quenching-based kinetic assay for DNA unwinding
These assays were performed in 60 µl helicase buffer with 20 nM DNA substrate (in molecules). The oligo F (49-mer) in DNA substrate was labelled with 6-flouroscein amidite (6-FAM) at the 5′ end whereas oligo R (22-mer) was labelled at 3′ end with rhodamine. The reactions were assembled on ice in 96-microwell plate and the recombinant proteins were directly added to their respective wells. The microplate was transferred to a microplate reader (CLARIOstar, BMG Labtech) at 37 °C and 6-FAM intensity was continuously monitored at every 30 s for 60 min.
The final values were plotted as graphs using GraphPad PRISM.
Electrophoretic mobility shift assay EMSA reactions (15 µl) were performed in binding buffer containing 25 mM Tris-HCl pH 8.0, 2 mM ATP, 2 mM MgCl 2 , 1 mM DTT, 50 mM NaCl, 0.1 mg ml −1 BSA (New England Biolabs) and 5′-end-FITC-labelled 25 nM DNA substrate (in molecules). All of the steps except for the assembling reactions and protein addition were performed in the dark. The reactions were assembled on ice and recombinant proteins were added to reactions, mixed and incubated for 10 min at 37 °C in the dark. Reactions were supplemented with 5 µl of EMSA loading buffer (50% glycerol, bromophenol blue) and resolved with 6% native TBE polyacrylamide gel (19:1 acrylamide-bisacrylamide, Bio-Rad) using the Mini-Protean Tetra Cell electrophoresis system (Bio-Rad) at 80 V for 45 min on ice. Finally, gels were imaged using the ChemiDoc MP imaging system. Scans of the gels are provided in the Supplementary Information.

RPA stripping gel-based assay
The stripping assay was performed as described for EMSA except that the products were resolved at room temperature and longer 6% TBE native gel was used.
DNA annealing assay DNA annealing assays were performed in 15 µl annealing buffer containing 25 mM Tris-HCl pH 8.0, 2 mM ATP, 2 mM MgCl 2 , 1 mM DTT, 50 mM NaCl and 0.1 mg ml −1 BSA (New England Biolabs). All of the steps except for the assembling reactions and protein addition were performed in the dark. For DNA substrate, 10 nM (in molecules) complementary oligos (5′-FITC-oligo 1 and oligo 2) were separately incubated in 7.5 µl annealing buffer and as indicated, with or without RPA on ice for 2 min. Recombinant proteins were added to FITC-oligo 1 reactions (7.5 µl) on ice, immediately followed by the addition of oligo 2 reactions (7.5 µl). Reactions were incubated for 10 min at 37 °C. The final concentration of both individual oligo and annealed dsDNA product was 5 nM. The reactions were stopped with 5 µl of 2% stop solution (0.2% SDS, 30% glycerol, 150 mM EDTA, bromophenol blue) and 1 µl proteinase K (Roche, 18.4 mg ml −1 ) and incubated for 10 min at 37 °C. To prevent the detection of spontaneous annealing during deproteination, 25-fold excess of unlabelled oligo 1 to FITC-oligo 1 was included in the 2% stop solution. The products were resolved and imaged identically as described for the unwinding assays.

Interaction assay
To study the interactions between HELQ and RAD51, MBP-HELQ-Flag baculovirus was expressed in 300 ml insect cells, and soluble extract from the collected pellet was prepared as described for the protein purification procedure. Reagent volumes for the preparation of the soluble extract were scaled down accordingly. The soluble extract was divided equally into two parts and incubated with amylose (E8021, NEB) and anti-Flag M2 resin (A2220, Sigma-Aldrich) for 1 h at 4 °C. Next, amylose resin and anti-Flag M2 resin were washed with wash buffer I (50 mM Tris-HCl pH 8.0, 1 mM DTT, 310 mM NaCl, 10% glycerol, 1 mM PMSF). Both resins were divided into 50 µl volumes in separates microtubes. 4 µg recombinant RAD51 was added to all except for one tube for each resin and incubated for 1 h at 4 °C. Resins were washed with Article wash buffer II (the same as wash buffer I but containing 100 mM NaCl). Proteins were eluted from resin in 1× SDS buffer by boiling at 95 °C for 4 min. The eluate was separated by 4-12% native SDS-PAGE gel (NuPAGE Bis-Tris, Invitrogen) and stained with instant blue Coomassie protein stain (Abcam).

DNA capture assay
The capture assays were performed in 20 µl DNA annealing buffer supplemented with 0.05% Tween-20. The reactions were assembled on ice and, where indicated, 82 nM RPA, 10 nM biotinylated dT43 (bio-dT43) and 10 nM 3′Cy3-dT79 were added to reactions. Next, HELQ was added to reactions as indicated. Reactions were mixed and incubated at 37 °C for 8 min in the dark. To pull-down bio-dT43, magnetic streptavidin beads were washed twice with PBS-0.1% Tween-20 (Dynabeads M-280, Thermo Fisher Scientific) and 5 µl of beads was added to each reaction. Reactions were further incubated for 4 min in the dark at room temperature and then washed twice with 80 µl washing buffer (25 mM Tris-HCl pH 8.0, 2 mM ATP, 2 mM MgCl 2 , 1 mM DTT, 100 mM NaCl, 0.5 mg ml −1 bovine serum albumin, NP-40) on a magnetic rack. Finally, the beads were resuspended in 30 µl loading buffer (7.5 µl 2% stop solution and 22.5 µl washing buffer) and boiled at 95 °C for 4 min. The samples were centrifuged at high speed for 1 min and 25 µl volume sample was loaded immediately on 10% native polyacrylamide gel and run as described for the unwinding assay. The gels were directly imaged in the ChemiDoc MP imaging system (Bio-Rad).

Substrate and flow cell preparation for SMI analysis
Experiments were performed using the commercially available C-trap (LUMICKS) set-up. Protein channels of the microfluidics chip were first passivated with BSA (0.1% (w/v) in PBS) and Pluronics F128 (0.5% (w/v) in PBS), with a minimum 500 µl of both flowed through before use. Biotinylated ssDNA precursor was prepared as described previously 33 . To generate gapped λDNA, a protocol described previously was used 9 . In brief, biotinylated hairpin oligonucleotides were annealed to λ-dsDNA ends and ligated 34 . Streptococcus pyogenes Cas9 D10A nickase (IDT) bound to previously described 16 guide RNAs was subsequently used to generate targeted DNA nicks. The reaction was then stored at 4 °C and directly diluted in PBS on the day of the experiment. DNA was captured between 4.5 µm SPHERO Streptavidin-coated polystyrene beads at 0.005% (w/v) using the laminar flow cell, stretched and held at forces of 100 pN (for ssDNA) or 65 pN (λ-gDNA 4/5) until the strands were fully melted. The presence of ssDNA and/or a ssDNA gap was verified by comparison with a built-in freely joined chain model. For confocal imaging, three excitation wavelengths were used-488 nm for eGFP and Alexa Fluor 488, 532 nm for Cy3 and 638 nm for Cy5, with emission detected in three channels with blue filter 512/25 nm, green filter 585/75 nm and red filter 640 LP.

Single-molecule DNA unwinding assay
For all the unwinding assays, the λ-gDNA 4/5 construct was held at a constant force of 50 pN. Beads and DNA were kept in PBS during the experiment, while DNA was melted in 0.5× NTM buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 mM MgCl 2 ) supplemented with 0.2 mg ml −1 BSA and 1 mM DTT. HELQ (50 nM) and/or 25 nM RAD51(A488) were flowed into the system in 1× HELQ buffer (25 mM Tris-HCl pH 8.0, 2 mM MgCl 2 , 50 mM NaCl) supplemented with 2 mM ATP, 0.2 mg ml −1 BSA and 1 mM DTT. Unwinding was monitored by the change in the distance between the beads over time. To directly image fluorescent RAD51, the following image acquisition set-up was used: 4 µW blue laser power, 0.1 ms px −1 dwell time, 100 nm pixel size, 1 s interframe wait time.

SMI-based RPA/RAD51 stripping
Using optical tweezers in the stripping assays, the λ-gDNA 4/5 construct was held at a distance corresponding to a force of 10 pN after melting. The beads and DNA were kept in PBS during the experiment (microfluidic channels 1 and 2), while DNA was melted in PBS (microfluidic channel 2) and subsequently incubated with 5 nM RPA-eGFP or a mixture of 100 nM RAD51 and 100 nM Alx-RAD51 in 1× HELQ buffer (25 mM Tris-HCl pH 8.0, 2 mM MgCl 2 , 50 mM NaCl), 0.2 mg ml −1 BSA and 1 mM DTT in channel 3. Once RPA-eGFP or Alx-RAD51 were assembled on λ-gDNA 4/5 (after ~2 min of incubation) beads with DNA were moved to channel 4 containing 50 nM HELQ in 1× HELQ buffer (25 mM Tris-HCl pH 8.0, 2 mM MgCl 2 , 50 mM NaCl) supplemented with 2 mM ATP, 0.2 mg ml −1 BSA and 1 mM DTT. RPA-eGFP or Alx-RAD51 signal disappearance was monitored over time. Image acquisition setup was performed as follows: 1.6 µW blue laser power, 0.1 ms px −1 dwell time, 100 nm pixel size, 1 s interframe wait time.

Image processing and data analysis
Real-time force, distance and fluorescence data were exported from Bluelake HDF5 files and analysed using custom scripts in the Pylake Python package. Force was downsampled to 3 Hz for plotting. The worm-like chain model for λ-dsDNA was used as a reference for forceextension curve comparison. Bead distance-time traces were processed in GraphPad Prism 7. First derivative and smoothing of the traces were performed to extract individual unwinding stroke rates. Unwinding stroke rate distribution was analysed in GraphPad Prism 7 by fitting a single or double Gaussian curve. Dwell times and binding frequencies were estimated in Fiji. Dwell-time frequency distribution was analysed in GraphPad Prism 7. Mann-Whitney U-tests were used to assess statistical significance of the data where appropriate.
For the position, velocity and mean square displacement (MSD) analysis (Extended Data Fig. 4i-k), we used a custom-made single-particle tracking algorithm in Python (https://github.com/singlemoleculegroup). The sub-pixel position of the fluorescent particle in each frame of the kymograph was calculated by fitting the signal intensity of a three-frame moving window with a 1D Gaussian function (linetime = 0.997 s, 100 nm px −1 ).
For the obtained trajectories, the MSD was calculated using the following equation: where N is the total number of frames in the kymograph, n is the number of frames within a moving window (lag time) from which the square displacement was calculated (ranging from 1 to N − 1) and X i is the molecule position along the DNA in time.
To evaluate whether a trajectory represents random walk or directed motion, the MSD dependency was fitted with a power law. A particle exhibits free or constrained diffusion with rate D when the MSD scales with an exponent α ≤ 1. When α > 1, the process is characterized as superdiffusive motion (for example, unidirectional walk).
To estimate the average velocity of the translocating molecule, the total route of the molecule (a sum of frame-to-frame displacements) was divided by the total trajectory time. Here, every trajectory was smoothed using the Savitzky-Golay filter (smoothing factor = 51) to eliminate tracking inaccuracies and the molecule's thermal fluctuations.
To estimate the loop size formed by the HELQ-RPA-DNA complex, the contour length after each unfolding event in the force-distance curve was fitted by the worm-like chain model. The difference between contour lengths of the neighbouring events corresponds with the loop size.
RPA stripping using single-molecule FRET Flow chambers were prepared as described previously 1,2 . Quartz slides and coverslips were passivated with polyethylene glycol (5% biotinylated) and flow chambers were constructed using double-sided sticky tape and sealed with epoxy. 5′-biotin-and internal amino linker-modified DNA oligonucleotides were labelled with Cy3-NHS ester and HPLC purified as previously described 3 . DNA (6 pM) was immobilized through biotinstreptavidin interactions. All of the experiments were performed in the standard HELQ buffer with addition of the PCA/PCD oxygen scavenger system with 5 mM PCA, 100 nM PCD and saturating Trolox. The flow chambers were imaged on a home-built, prism-based total internal reflection microscope with a 532 nm excitation laser (~3.8 mW), and images were acquired on an EM-CCD camera (Andor) with a 30 ms exposure time. FRET efficiencies were calculated from integrated donor (I D ) and acceptor (I A ) intensities as FRET = I A /(I D + I A ) (refs. 1,3 ). The images and data were analysed using custom IDL, MATLAB and R scripts, which are available on request. FRET efficiency histograms were constructed by averaging the first 10 frames of each trajectory, with bins of 0.1. The dwell times of the free (high FRET) and bound (low FRET) states were measured, and dwell-time histograms were plotted. These were fit with single exponential fits to obtain average dwell times.

Cell culture
The U2OS human osteosarcoma cell line was grown in DMEM supplemented with 10% bovine growth serum, 2 mM l-glutamine, 100 µg ml −1 streptomycin and 100 U ml −1 penicillin. U2OS-EJDR cells were cultured in DMEM supplemented with 10% tetracycline-free fetal bovine serum, 2 mM l-glutamine, 100 µg ml −1 streptomycin and 100 U ml −1 penicillin. U2OS-DR cells contain a stably integrated DR-GFP reporter to measure DSB repair by HR as previously described 35 . U2OS-SA cells contain a stably integrated SA-GFP reporter to measure DSB repair by SSA as previously described 36 . U2OS-DR cells containing a stably integrated EJ-RFP reporter to measure DSB repair by mutagenic end-joining constitute the U2OS-EJDR cell line as previously described 37 . U2OS-RFP-SCR cells contain a stably integrated RFP-SCR reporter for quantifying STGC and LTGC in HR as previously described 38 .

DSB repair assays
Cells (0.25 × 10 6 ) were reverse-transfected with 30 pmol siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, cells were transfected with 2 µg of pCMV-ISceI-3×NLS or pCMV 3×NLS empty vector and 30 pmol siRNA using Lipofectamine 2000 (Invitrogen). Cells were collected for analysis by flow cytometry at 72 h using the LSR Fortessa instrument (BD Biosciences). For each experiment, the percentage of GFP-or RFP-positive cells in the empty vector control was subtracted from the I-SceI-transfected cells. Data from each reporter assay represent the mean ± s.e.m. of at least three independent experiments, and statistical analysis was performed using two-tailed paired t-tests. The Cas9-mediated DSB repair assay was used to introduce a Cas9-mediated site-specific DSB and break repair outcomes were detected using next-generation sequencing as previously described 39 . In brief, cells were transfected with AAVS1 T2 CRISPR in pX330 (Addgene plasmid, 72833) using Lipofectamine 3000 (Invitrogen) and collected for genomic DNA extraction after 48 h. To measure SSTR, 1 µl of a 10 µM 121 bp donor oligonucleotide with three substitutions (purchased from IDT) was co-transfected with the CRISPR plasmid. A 201 bp PCR amplicon covering the expected Cas9 break site was sent for next-generation sequencing and reads were analysed for insertions, deletions and substitutions. NHEJ is defined as 1-5 bp deletion, MMEJ as >5 bp deletion with at least 2 bp microhomology, and SSTR as the introduction of three 1 bp substitutions. Data represent the mean ± s.e.m. of at least four independent experiments, and statistical analysis was performed using two-tailed paired t-tests.

Immunoprecipitation and western blot
Cells were lysed in RIPA buffer (Teknova, R3792) with HALT protease inhibitor cocktail (Thermo Scientific Scientific). As HELQ is expressed at low levels in human cell lines and the commercially available antibodies tested did not dependably detect endogenous HELQ by western blot, we validated siRNA-mediated HELQ knockdown using HELQ immunoprecipitation. Cells (10 6 ) were transfected with 2 µg siRNA by electroporation using the Amaxa nucleofector system, and plated into 150 mm dishes. After 72 h, cells were collected and whole-cell extracts were used for immunoprecipitation. Protein (2 mg) was incubated with 1 µg anti-HEL308 antibodies (Novus Biologicals, NBP1-91842) at 4 °C overnight with rotation. After washing 0.25 mg Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, 88802), the antigen sample-antibody mix was added to the beads and incubated for 1 h at room temperature with rotation. The beads were washed four times and eluted in SDS-PAGE reducing sample buffer (Invitrogen) for 10 min at 96 °C. The samples were loaded onto 4-12% Bis-Tris precast gels (Invitrogen) for SDS-gel electrophoresis and transferred onto Immobilon-P PVDF membrane (Millipore). Membranes were blocked for 1 h at room temperature with Pierce clear milk blocking buffer. For western blot analysis of RAD52 and BRCA2, 50 µg of protein was loaded onto 10% Bis-Tris or 3-8% Tris-acetate precast gels (Invitrogen) for SDS-gel electrophoresis. Proteins were transferred onto a PVDF (polyvinylidene difluoride) membrane (BioRad) for BRCA2 detection. The iBlot Gel Transfer System (Invitrogen) was used to perform dry blotting of proteins onto nitrocellulose membranes for RAD52 detection. Membranes were blocked for 1 h at room temperature with Pierce clear milk blocking buffer. Antibodies for the western blot analysis were as follows: anti-HEL308 (Santa Cruz Biotechnology, sc-81095), anti-RAD52 (Santa Cruz Biotechnology, sc-365341), anti-BRCA2 Ab-1 (Millipore Sigma-Aldrich, OP95), anti-SMC1 (Bethyl laboratories, A300-055A).

Gene expression
Cells were collected 72 h after siRNA transfection. Cell lysis, reverse transcription and quantitative PCR were performed using the TaqMan Gene Expression Cells-to-CT kit from Invitrogen according to the manufacturer's instructions. TaqMan Gene Expression Assays for HELQ (Hs01127906_m1) and ACTB endogenous control (Hs99999903_m1) were run in triplicate on the QuantStudio 6 Pro real-time PCR instrument (Applied Biosystems). Relative fold gene expression was calculated using the ∆∆C t method. Data represent the mean ± s.e.m. of at least three independent experiments, and statistical analysis was performed using two-tailed paired t-tests.

Article
Immunofluorescence WT U2OS-DR and HELQ −/− U2OS-DR cells were seeded onto four-chamber tissue culture slides (Millipore) and treated the next day with 4 µM camptothecin or 10 Gy irradiation. For siRNA knockdown, cells were transfected with non-targeting or HELQ siRNA, incubated overnight and seeded onto chamber slides the next day. Cells were treated with the designated damaging agent 48 h after siRNA transfection. At the designated time points, cells were fixed, blocked and permeabilized. Cells were stained with the following antibodies: anti-phosphorylated-histone H2A.X (Ser139) (05-636, Millipore Sigma-Aldrich), anti-RAD51 (PC130 Millipore Sigma-Aldrich), anti-RPA32 (2208, Cell Signaling Technology), anti-phosphorylated-RPA32 (S4/S8) (ab87277, Abcam). Secondary antibodies were as follows: Alex-aFluor 488-labelled goat anti-rabbit IgG, AlexaFluor 568-labelled donkey anti-mouse IgG, AlexaFluor 568-labelled goat anti-rat (Invitrogen). Images were obtained using a Zeiss LSM 880 confocal laser scanning microscope and analysed using ImageJ. At least 100 nuclei were counted per experiment and nuclei with >5 foci were scored as positive. Data represent the mean ± s.e.m. of at least three independent experiments, and statistical analysis was performed using two-tailed paired t-tests.
The quantification of gel-based data was carried using ImageJ (NIH v.1.52k). Mars Data analysis software (BML Labtech v.3.10 R6) was used for quenching-based kinetic unwinding assays. To analyse the SMI by optical tweezer, Pylake software from Lumicks was used. Furthermore, custom scripts were used for analysing some of unwinding/translocation assays carried out by SMI (https://github.com/singlemoleculegroup). smFRET analysis was performed using iSMS software (open source) 39 . For microscopy data analysis, Image J (NIH, v.1.53e) was used. QuantStudio Design and Analysis Software v.2 was used with the Quant-Studio 6 Pro real-time PCR instrument for relative gene expression analysis. Flow cytometry data were analysed using BD FlowJo (v.10.6.2). FlowJo was used to gate single cells and then select for GFP + and/or RFP cells depending on the assay as shown. The background signal from the untransfected control was subtracted from each experiment. Representative plots from FlowJo showing the gating strategy are provided in Supplementary Fig. 2. Cas9 DSB repair assay sequencing data were analysed as in Hussain et al. 40 , using PEAR software for read stitching, BLOSUM62 for alignment and code for microhomology/deletion analysis available on GitHub (https://github.com/cjsifuen/delmh). Statistical analysis was performed using GraphPad Prism (v.8.2.1 and v.8.4.2). All schematics except for those of the quenching-based kinetic unwinding assays were generated using Adobe illustrator v.2.3. For the schematics of quenching-based kinetic unwinding assays, Biorender.com was used.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
The datasets generated during and/or analysed during the current study are included alongside the Article. Raw datasets for experiments performed on the C-TRAP are not included and are available from S.J.B. on reasonable request. Gel source data are provided in the Supplementary Information. All data are archived at the Francis Crick Institute or Sloan Kettering Institute.

Code availability
The custom script made for determining the DNA unwinding/translocation by SMI is available at GitHub (https://github.com/singlemoleculegroup).

Corresponding author(s):
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Data analysis
We used combination of custom made, commercial or publicly available software used for data analysis. These include ImageJ (NIH Version 1.52k) for quantifying gel based assays, Mars Data analysis software (BML Labtech version 3.10 R6), Pylake software (Lumicks) for singlemolecule imaging by optical tweezer, Custom script made for some of DNA unwinding/translocation SMI assay (https://github.com/ singlemoleculegroup), iSMS software (open source) for smFRET analysis (Preus et al. iSMS: single-molecule FRET microscopy software. Nat Methods 12, 593-594 (2015)), Image J (NIH, Version 1.53e) for the analysis of microscopy data; graphs and numerical data (including statistics/error bars) was analyzed and plotted by Prism (GraphPad, Version 8.2.1 and 8.4.2); flow cytometry data was analyzed using FlowJo v10.6.2; QuantStudio Design and Analysis Software v2 was used with QuantStudio 6 Pro real-time PCR instrument for relative gene expression analysis; Cas9 DSB repair assay sequencing data was analyzed as in Hussain et al, Nucleic Acids Research 2021, using PEAR software for read stitching, BLOSUM62 for alignment, and code for microhomology/deletion analysis available on Github https://github.com/cjsifuen/delmh. For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The datasets generated during and/or analysed during the current study are included alongside the Article or are available from S.J.B and S.P on reasonable request. For gel source data, see source data file. All data are archived at the Francis Crick Institute or Sloan Kettering Institute. The custom script made for determining the DNA unwinding/translocation by SMI is available at link "https://github.com/singlemoleculegroup".

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Replication
To ensure the replication, each experiment was performed multiple times in exact condition on different days. For protein-protein interaction, interaction assay was repeated thrice with slightly different conditions and all showed the same result indicating the robustness of the result. Some gels were not included in the study due to technical failure. Any result, which did not replicate was not included in the study.
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