Cells
We maintained Expi293F cells (Thermo Fisher Scientific, A14527) in Expi293 Expression Medium (Thermo Fisher Scientific, A1435101) according to the manufacturer’s instructions. The absence of mycoplasma contamination was verified using the e-Myco PCR detection kit (Bulldog Bio, 25234), with testing performed monthly.
Protein expression and purification
All MPXV protein sequences were derived from MPXV isolate hMPXV/P12/2022 (clade IIb). The sequence encoding full-length MPXV E5 (GenBank: XNX20584.1, residues 1–785), E5(ΔRRM) (GenBank: XNX20584.1, residues 238–785), F8 (GenBank: XNX20538.1, residues 1–1006), F8 with a C-terminal deletion (GenBank: XNX20538.1, residues 1–984) or E4 (GenBank: XNX20583.1, residues 1–218) was cloned into the pCAGGS vector containing a maltose-binding protein (MBP) at the N terminus followed by HRV 3C cleavage site (LEVLFQGP). The sequence encoding wild-type A22 (GenBank: XNX20615.1, residues 1–426) or A22 with the F217A and F263A substitutions (GenBank: XNX20615.1, residues 1–426) were cloned into a pCAGGS vector without tag. Plasmids encoding MPXV polymerase subunits (F8, A22 and E4) were co-transfected into Expi293F cells maintained in suspension using polyethylenimine (PEI) (25000 MW, Polysciences), and E5 was transfected into Expi293F cells grown in suspension using PEI (25000 MW, Polysciences) when the cells reached a density of 2 × 106 cells per ml.
For wild-type MPXV polymerase holoenzyme (F8–A22–E4) and MPXV polymerase holoenzyme mutant purification, after culturing at 37 °C for 72 h, cells were collected by centrifugation at 4,000g for 20 min. Cells were lysed using lysis buffer containing 50 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and protease inhibitor (cOmplete, Mini, EDTA-free protease inhibitor cocktail, Millipore Sigma, 11836170001). Cell debris was removed through centrifugation 50,000g for 2 h on the Ti50.2 rotor. The supernatant was incubated with amylose resin (NEB, E8021S) at 4 °C for 1 h and washed with wash buffer (25 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA and 1 mM DTT). Bound MPXV F8–A22–E4 complex was subjected to on-column digestion overnight with HRV-3C protease (TaKaRa, 7360) at 4 °C. The eluate fractions within the elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT) were concentrated to 500 μl for further purification using Superdex 200 increase column (Cytiva, 28990944). Fractions containing MPXV polymerase holoenzyme were pooled, concentrated to about 1.5 μg μl−1 and stored for the further structural and functional studies. For exonuclease-defective polymerase mutant, residues Asp166 and Glu168 in F8 were substituted to alanine using site-directed mutagenesis of the wild-type F8 pCAGGS vector, and the same purification strategy was used to purify the polymerase holoenzyme.
For MPXV E5 or E5(ΔRRM) purification, after culturing at 37 °C for 72 h, cells were collected by centrifugation at 4,000g for 20 min. Cells were lysed using lysis buffer containing 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and protease inhibitor (cOmplete, Mini, EDTA-free protease inhibitor cocktail, Millipore Sigma, 11836170001). Cell debris was removed through centrifugation 50,000g for 2 h with Ti50.2 rotor. The supernatant was incubated with amylose resin (NEB, E8021S) at 4 °C for 1 h and washed with wash buffer (25 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA and 1 mM DTT). Bound MPXV E5 or E5(ΔRRM) were subjected to on-column digestion overnight with HRV-3C protease (TaKaRa, 7360) at 4 °C. The eluate fractions with the elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT) were concentrated to 1 ml for further purification using the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV E5 or E5(ΔRRM) hexamers were pooled, concentrated to about 3 μg μl−1 and stored for the further structural and functional studies. For the primase-dead E5 mutant, Asp70 in E5 was substituted to alanine using site-directed mutagenesis of the wild-type E5 pCAGGS vector, and the same purification strategy was used. All purified proteins and complexes were analysed using SDS–PAGE.
MPXV replisome assembly
To determine the structure of the ssDNA-bound MPXV replisome, we used an 80 nucleotide DNA template (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGCTCCCGCGTCGGAGTCGTTTCGACTCCGACGCGGGAGC-3′) (template 1) (Extended Data Fig. 1c and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and DNA were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT overnight. The mixture was applied onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent analyses.
To determine the structure of the forked DNA-bound MPXV replisome, we used an 83 nucleotide DNA template (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTCGGAGTCGTTTCGACTCCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′) (template 2) (Extended Data Fig. 5b and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and DNA were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT overnight. The mixture was applied onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent analyses.
To determine the structure of the MPXV replisome bound to RNA–DNA hybrid, we used a 58 nucleotide DNA oligo (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCCCGCGTCGGAGTCG-3′) and an 18 nucleotide RNA oligo (5′-CGACUCCGACGCGGGAGC-3′) (template 3) (Extended Data Fig. 9b and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and RNA–DNA hybrid were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT with overnight incubation. The mixture was passed onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent structural analysis.
Cryo-EM sample preparation and data collection
Samples in the elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT) were vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific), with samples maintained at 100% humidity at room temperature. We applied 4 µl of sample to Quantifoil Au 1.2/1.3 300 mesh (EMS Q450CR1.3) grids that were previously plasma treated in a PELCO easiGlow discharge cleaning system at 0.39 mBar, 15 mA, for 30 s and used blot times of 3 s.
We collected datasets using EPU (v.2.13) on a Titan Krios (Thermo Fisher Scientific) operating at 300 kV and a Falcon 4 detector with Selectris energy filter (Thermo Fisher Scientific) in counting mode at ×165,000 magnification, corresponding to calibrated pixel size of 0.74 Å px−1. For the ssDNA-bound (template 1) MPXV replisome dataset, 20,997 micrographs were collected at a dose rate of 8.88 e– px−1 s−1. The total exposure time of 3.28 s was divided into 56 frames (total dose of approximately 54 e− Å−2). For the forked DNA-bound (template 2) MPXV replisome dataset, 45,400 micrographs were collected with the pixel size of 0.74 Å px−1 at a dose rate of 10.92 e− px−1 s−1. The total exposure time of 2.55 s was divided into 87 frames (total dose of approximately 51 e− Å−2). For the structure of the MPXV replisome bound to RNA–DNA hybrid (template 3), 27,692 micrographs were collected with the pixel size of 0.74 Å px−1 at a dose rate of 11.02 e− px−1 s−1. The total exposure time of 2.52 s was divided into 43 frames (total dose of approximately 51 e− Å−2).
Cryo-EM data processing
We performed all image processing using Relion 3.0 (v.3.1.4)47 and cryoSPARC (v.4.4.1)48. For datasets collected on the MPXV replisome with DNA, video frames were gain-normalized and motion-corrected using MotionCor2 (v.1.5.0)49. Contrast transfer function (CTF) correction was performed using CTFFind4.1 (v.4.1.14)50, as implemented in Relion 3.0.
For the ssDNA-bound MPXV replisome, we performed automated particle picking with 4,073,693 particles (Extended Data Fig. 1d). After several rounds of two-dimensional (2D) classification, a subset of particles (186,468 in total) generated from the first round of heterogeneous refinement of 1,210,465 particles was subjected to second-round heterogeneous refinement. A subset of particles (81,800 in total) generated from the second round of heterogeneous refinement was subjected to a third round 3D classification, and a subset of particles (56,874 in total) generated from the 3D classification was processed for homogeneous refinement, yielding a final map of 4.1 Å resolution. To obtain higher-resolution maps for MPXV replisome, we masked the E5 helicase module and polymerase–primase module for further local refinement, yielding the final maps of 3.8 Å and 3.5 Å with improved density, respectively (Extended Data Fig. 1d–g). After several rounds of 2D classification, a subset of particles (30,240 in total) generated from the first round of heterogeneous refinement of 1,210,465 particles was subjected to local refinement, yielding a MPXV polymerase–primase core complex map of 4.0 Å resolution (Extended Data Fig. 1h).
For the forked DNA-bound MPXV replisome, after several rounds of 2D classification, a subset of particles (31,005 in total), which had been generated from the second round of heterogeneous refinement of 175,757 particles, was subjected to homogeneous refinement, yielding a final map of 6.5 Å resolution (Extended Data Fig. 5a). To obtain higher-resolution maps for forked DNA-bound MPXV replisome, we masked the E5 helicase module and polymerase–primase module for further local refinement, yielding the final maps of 4.5 Å and 4.1 Å with improved density, respectively (Extended Data Fig. 5c–e).
For the structure of the MPXV polymerase–primase core complex bound to RNA–DNA hybrid, after several rounds of 2D classification, a subset of particles (25,351 in total), which had been generated from the second round of heterogeneous refinement of 61,436 particles, was subjected to homogeneous refinement, yielding a final map of 3.8 Å resolution (Extended Data Fig. 9c,d).
Model building, refinement and figure generation
Two previous cryo-EM structures of ssDNA-bound E5 hexamers differed in the polarity of the ssDNA within E5 (refs. 3,4). Of these previous structures, to aid with initial model building, we used the higher-resolution structure (PDB: 8XJ7)4, as the ssDNA polarity was consistent with that observed in other superfamily-3 helicases51,52. We also used the cryo-EM structure of MPXV polymerase (PDB: 8HG1)5. These were fitted as initial models into cryo-EM map using UCSF Chimera X (v.1.2)53. E5B, E5C, E5D and E5E residues 1–323, which comprise the primase domain of E5, could also not be visualized in cryo-EM maps of the complex. To build the model of the ssDNA-bound replisome, we used the 3.5 Å map with mask focused on the polymerase–primase region to build the model of polymerase, E5A primase and E5F RRM. The E5F RRM had weak density but we could nonetheless dock the corresponding coordinates from the E5 cryo-EM structure (PDB: 8HWA)3 based on clear density for the α-helices and β-sheets. For E5 helicase, we used the 3.8 Å helicase-focused map for model building. For the interface between E5A ZBM and E5 helicase, we used the 4.1 Å overall map for model building. For the forked DNA-bound MPXV replisome, we used the structure we generated of the ssDNA-bound replisome, which was from higher-resolution maps, as a starting point for model building. For the polymerase–primase core complex bound to RNA–DNA hybrid, we used the 3.8 Å map for model building and rigid body docking, respectively. We performed manual adjustment and iterative model building and real space refinement using Coot (v.0.9.8.8)54 and PHENIX (v.1.21-5207)55. Figures were generated using PyMol (v.2.5.4) and UCSF Chimera X (v.1.2)53. For the structure of the polymerase–primase core complex bound to RNA–DNA hybrid, a longer segment (including 4 bp) for which density could be modelled but was too poor to be deposited was used for figure generation.
MP analysis
MP analyses were carried out using the Refeyn TwoMP mass photometer (Refeyn) at room temperature. Glass coverslips and gaskets were cleaned with HPLC-grade water and isopropanol and dried under filtered gas before use. MPXV E5 alone, polymerase alone, E5 and polymerase with or without DNA substrate and E5 and polymerase containing mutant 2 or 3 subunits were diluted to 200 nM in elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM DTT). Next, 18 μl of buffer was used to find the camera focus before loading 2 μl of the sample onto the gasket. The acquisition camera image size was set to medium. Data were collected as a 1 min video and then processed using ratiometric imaging. To correlate ratiometric contrast to mass, the Refeyn TwoMP instrument was calibrated using molecular standards of monomeric BSA (66 kDa), dimeric BSA (132 kDa) and thyroglobulin (660 kDa) with a molecular mass error of less than 5%. Data were analysed using DiscoverMP v.2.3 (Refeyn).
Helicase activity assay
For helicase activity assays with DNA containing a 5′ overhang, the oligonucleotides 5′-TTTTTTTTTTAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3′ and 5′-AGCTATGACCATGATTACGAATTGCTTAATTCGTGCAGGCATG-3′ with 5′-labelled 6-FAM were annealed to generate the 5′-overhang-containing DNA substrate (Supplementary Table 2). Helicase assays with 6-FAM-labelled substrates (50 nM) and E5 (0.2, 0.5, 1 and 1.5 μM) were performed in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP, which was named the E5-only group. For E5-polymerase group, assays were performed with 6-FAM-labelled substrates (50 nM), E5 (1 μM) and polymerase (0.5, 1 and 2 μM) in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP. The E5-only group and E5–polymerase group were incubated at 4 °C for 1 h, respectively, and then were incubated at 37 °C for 2 h. To terminate reactions, we added 20 mM EDTA, 0.5% (v/v) SDS, 0.2% (v/v) bromophenol blue and 2 μM of an unlabelled DNA strand and transferred to 4 °C for 30 min, and proteins were digested by 2 mg ml−1 proteinase K (Roche, 3115887001) at room temperature for 30 min. Products were separated on a 20% polyacrylamide–TBE gel, and gels were exposed to Typhoon FLA 9500 (GE Healthcare) for imaging. We used Image Studio Lite (v.5.2) for gel quantification. To quantify relative ssDNA products, a box of equivalent size to that used for other bands of the same template was drawn at the ssDNA position on the negative-control lane and used to set the background to 0. The resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM E5 with 2 μM polymerase), which was set to 1.
For helicase activity assays with forked DNA substrate, the oligonucleotides 5′-TTTTTTTTTTTTTTTTTTTTAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT with 3′-labelled 6-FAM and 5′-AGCTATGACCATGATTACGAATTGCTTAATTCGTGCAGGCATGTTTTTTTTTTTTTTTTTTTT-3′ were annealed to generate the forked DNA substrate (Supplementary Table 2). Helicase assays with 6-FAM-labelled substrates (50 nM) and E5 (0.5, 1 and 1.5 μM) were performed in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP, which was named the E5-only group. For the E5–polymerase group, assays were performed with 6-FAM-labelled substrates (50 nM), E5 (1 μM) and polymerase holoenzyme (0.5, 1 and 2 μM) in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP. The E5-only group and E5–polymerase group were incubated at 4 °C for 1 h and were then incubated at 37 °C for 2 h. For all reactions under various conditions, 20 mM EDTA, 0.5% (v/v) SDS and 0.2% (v/v) bromophenol blue were added to terminate the reaction, and the samples were transferred to 4 °C for 30 min. Products were separated on a 20% polyacrylamide–TBE gel, and gels were exposed to Typhoon FLA 9500 (GE Healthcare) for imaging. We used Image Studio Lite (v.5.2) for gel quantification. To quantify relative ssDNA products, a box of equivalent size to that used for other bands of the same templates was drawn at the ssDNA position on the negative-control lane and used to set the background to 0. The resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM E5 with 2 μM polymerase), which was set to 1.
The helicase assays evaluating the effect of polymerase–E5 interface mutations were performed with a DNA substrate containing a 5′ overhang. The oligonucleotides 5′-TTTTTTTTTTAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT and 5′-AGCTATGACCATGATTACGAATTGCTTAATTCGTGCAGGCAT G-3′ with 5′-labelled 6-FAM were annealed to generate the 5′-overhang DNA substrate (Supplementary Table 2). Helicase assays were performed with E5 alone (1 μM), E5 (1 μM) with WT polymerase (2 μM) and E5 with polymerase containing mutant 1, 2 or 3 (2 μM) in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP. The helicase assays comparing wild-type E5 with E5(ΔRRM) were performed with a DNA substrate containing a 5′ overhang and E5 alone (1 μM), E5 (1 μM) with WT polymerase (1 μM) and E5(ΔRRM) (1 μM) in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP. All of the reactions were incubated at 4 °C for 1 h and then were incubated at 37 °C for 2 h. The time-course helicase assays were performed with E5 (1 μM) with polymerase (2 μM) using 5′-overhang DNA substrate or forked DNA in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP and 1 mM dNTP. To terminate reactions, we added 20 mM EDTA, 0.5% (v/v) SDS, 0.2% (v/v) bromophenol blue and 2 μM of an unlabelled DNA strand and transferred to 4 °C for 30 min. The protein was digested by 2 mg ml−1 proteinase K (Roche, 3115887001) at room temperature for 30 min. Products were separated on a 20% polyacrylamide–TBE gel, and gels were imaged on the Typhoon FLA 9500 (GE Healthcare) system. We used Image Studio Lite (v.5.2) for gel quantification. To quantify relative ssDNA products, a box of equivalent size to that used for other bands of the same templates was drawn at the ssDNA position on the negative-control lane and used to set the background to 0. For helicase assays with polymerase mutants, the resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM E5 with 2 μM wild-type polymerase), which was set to 1. For time-course helicase assays using 5′-overhang DNA substrate and forked DNA, the resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM E5 with 2 μM wild-type polymerase using 5′-overhang DNA substrate at 60 min), which was set to 1. For helicase assays with E5(ΔRRM), the resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM E5(ΔRRM)), which was set to 1. We used GraphPad Prism (v.10.1.2) for figure generation and statistical analysis of biochemical assays.
Polymerase DNA elongation assay
The oligonucleotides 5′-TTTTTTTTTTTTTTTTTTTTAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3′ and 5′-AGCTATGACCATGATTACGAATTGCTTAATTCGTGCAGGCATG-3′ with 5′-labelled 6-FAM were annealed to generate the 5′-overhang-containing DNA substrate (Supplementary Table 2). Elongation assays were performed with polymerase alone (1 μM), polymerase (1 μM) with WT E5 (1 μM) and polymerase (1 μM) with E5(ΔRRM) (1 μM) in 25 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2 and 5 mM dNTP. The reaction was incubated at 4 °C for 1 h and then was incubated at 37 °C for 2 h. All reactions under various conditions were added 20 mM EDTA, 0.5% (v/v) SDS and 0.2% (v/v) bromophenol blue to terminate the reaction and transferred to 4 °C for 30 min. The protein was digested by 2 mg ml−1 proteinase K (Roche, 3115887001) at room temperature for 30 min. Products were separated on a 20% polyacrylamide–TBE gel, and gels were exposed to Typhoon FLA 9500 (GE Healthcare) for imaging. We used Image Studio Lite (v.5.2) for gel quantification. To quantify DNA polymerase elongation activity, a box of equivalent size to that used for other bands of the same templates was drawn at the dsDNA position on the negative-control lane and used to set the background to 0. The resulting values were then normalized to the corresponding value from the lane in which the most positive result was obtained (1 μM polymerase alone), which was set to 1.
Optical tweezer experiments
Single-molecule experiments were performed on a C-trap (LUMICKS) integrating optical tweezers and microfluidics. The five-channel laminar flow cell was used for experiments after passivation using 0.5% (w/v) Pluronic F127 in PBS, and subsequently with BSA (1 mg ml−1).
We performed the experiment with wild-type E5 and exonuclease-defective MPXV polymerase (F8–A22–E4). Streptavidin-coated polystyrene beads (0.005% (w/v); 4.35 µm, Spherotech, SVP-40-5) were injected into channel 1. Biotin-labelled 17 kb DNA molecule containing two nicks (5 kb apart) on one of the strands (about 2 pM) (LUMICKS, 00027) was flowed into channel 2. Buffer A, containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 0.1 mg ml−1 BSA and 1 mM DTT, was injected into channel 3. E5 was diluted to 10 nM in buffer A and injected into channel 4. In the end, E5 and polymerase (exonuclease deficient) were diluted to 10 nM and 20 nM in buffer A, respectively, and injected into channel 5.
We conducted single-molecule experiments using wild-type E5 and an exonuclease-deficient polymerase on a five-channel microfluidic chip, loading reagents sequentially as follows: streptavidin-coated polystyrene beads (0.005% (w/v), 4.35 µm; Spherotech) were introduced into channel 1, a biotin-labelled 17 kb DNA substrate containing two nicks spaced around 5 kb apart on one strand (around 2 pM; Lumicks) was flowed into channel 2, and buffer A (25 mM HEPES, pH 7.5, 150 mM NaCl, 0.1 mg ml−1 BSA, 1 mM DTT) was loaded into channel 3 for baseline establishment and subsequent washing/equilibration; channels 4 and 5 were then used for protein delivery, whereby E5 alone (10 nM), polymerase alone (20 nM), E5 (10 nM) + polymerase (20 nM), E5 (10 nM) + polymerase (100 nM), and an E5(ΔRRM) (10 nM) were alternately introduced into the same channel(s) across measurements, and whenever switching between different protein conditions within a given channel, the system was extensively flushed with buffer A and re-equilibrated to minimize carryover and ensure consistent assay conditions.
All experiments were performed at room temperature (28 °C). The optical traps were calibrated using power spectrum of Brownian motion of the trapped beads to achieve a trap stiffness of 0.16–0.18 pN nm−1. After optically trapping two beads, the DNA molecule was tethered between the beads under flow in channel 2. The presence of a single DNA tether was verified by measuring a force–extension curve at a constant pulling rate of 0.2 µm s−1 and comparing it to worm-like chain model of dsDNA. The tethered DNA molecule was extended beyond the contour length of the DNA (6.34 μm) to around 8.5 μm and held for 10 s in the presence of flow to melt away a piece of ssDNA leaving behind a gap of 5 kb on the tethered DNA molecule. Subsequently, the DNA tether was moved to the protein channel (channel 5) and incubated for 10–30 s. In most of the experiments, this loading step was performed using DNA held at very low force (~1 pN). After protein loading, unwinding experiments were performed in the same channel under a constant force of 52 pN, with the resulting changes in distance between beads recorded using BlueLake software (v.2.6.4) for subsequent analysis. To ensure reproducibility across different conditions, the experiments were repeated for a total of 33 times for the E5:polymerase 1:2 ratio; 19 times for the E5:polymerase 1:10 ratio; and 25 times for the E5(ΔRRM) mutant (Supplementary Figs. 1d and 2a,b). Notably, measurements conducted with an E5:polymerase ratio of 1:2 at lower forces of 5 pN and 20 pN (n = 12 and 10, respectively) yielded no detectable unwinding activity, and measurements conducted with the E5(ΔRRM) mutant at 5 pN and 20 pN (n = 8 and 9, respectively) yielded weak unwinding activity (Supplementary Figs. 2c,d and 3).
To confirm the mechanical stability of the substrate, we performed control experiments at 52 pN in the absence of protein (Supplementary Fig. 1a). These measurements showed no evidence of spontaneous DNA unwinding or mechanical rupture, confirming that the observed activity was protein dependent. Moreover, we conducted controls using E5 alone (n = 19) and polymerase alone (n = 12) (Supplementary Fig. 1b,c), neither of which showed processive unwinding comparable to the MPXV E5 with polymerase or the E5(ΔRRM) protein alone.
Data acquisition and analysis
All data analysis was carried out in Python using custom-written scripts and Lakeview (v.1.3). Helicase activity, recorded as the change in distance between optically trapped beads, was converted to the number of nucleotides unwound42. From each raw trace, multiple 15 s windows (around 250 datapoints per window) were extracted. These segments were smoothed using a Savitzky–Golay filter, after which a linear regression was performed on each window to estimate the unwinding rates. To identify pause states, the raw data were first filtered using the Savitzky–Golay filter to reduce noise. The discrete derivative of the unwound nucleotides with respect to time was then computed. A threshold of 2 nucleotides per second was used. The timepoints with instantaneous unwinding rates below this threshold were classified as paused. The first 60 s of the unwinding trace was used for quantification of paused state duration (Supplementary Fig. 1e). Statistical comparisons between experimental conditions were performed using Welch’s t-tests. Pairwise comparisons were conducted as indicated in the text. All statistical analyses were implemented in Python using standard scientific libraries.
E5 primase RNA synthesis assay
To measure nucleotide synthesis, 2 µM of WT E5 and 4 µM or 0.02–4 µM of WT polymerase holoenzyme were incubated with 15 µM of M13 ssDNA (NEB, N4040S) on ice for 60 min. To measure nucleotide synthesis inhibition by mutant E5 or polymerase, 2 µM of E5(D70A) (primase dead) or E5(ΔRRM), and 10 µM WT, mutant 2 or mutant 3 polymerase holoenzyme were incubated with 15 µM of M13 ssDNA on ice for 60 min. The protein–DNA mixture was incubated with unlabelled 1,000 µM ATP (Jena Bioscience), 250 µM GTP, 250 µM UTP, 25 µM CTP (New England Biolabs, N0450L) and 0.5 µM α32P-labelled CTP (Perkin Elmer/Revvity, approximately 1 µCi) in reaction buffer (25 mM Tris-HCl pH 7.5, 30 mM KCl, 10 mM MgCl2, 10 mM DTT, 100 µg ml−1 BSA) at 37 °C for 60 min. Reaction mixtures (6 µl) were boiled at 80 °C for 10 min and terminated with addition of 0.5 µl of Quick CIP phosphatase (New England Biolabs, M0525S) at 37 °C for 90 min to remove terminal phosphate groups from unreacted nucleotides. Where indicated, reactions were additionally treated with 1 µl DNase I (Thermo Fisher Scientific, EN0521) or 1 µl RNase T1 (Thermo Fisher Scientific, AM2283) and 1 µl RNase A (NEB, T3018L). To visualize the ladder, 25 µM ssRNA (IDT) with scrambled sequence of varying nucleotide lengths (10, 15, 20, 25, 30, 40, 50, 60 nucleotides) was labelled with 10 U of T4 PNK (NEB, M0201L), 0.5 µM γ32P-labelled ATP (Perkin Elmer/Revvity, approximately 20 µCi) in reaction buffer (70 mM Tris-HCl pH 7.6, 10 mM MgCl2, 5 mM DTT) at 37 °C for 60 min. Each reaction was denatured with 2× STOP loading dye (95% (v/v) deionized formamide, 20 mM EDTA, 0.01% (v/v) bromophenol blue and xylene cyanol) and analysed on a 20% urea–PAGE gel and exposed to a phosphor-screen before imaging on the Typhoon Trio Variable Mode Imager (GE Healthcare).
Reporting summary
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