M13mp18

Oligonucleotides were purchased from Integrated DNA Technologies (IDT) with standard desalting, and the full sequences of all strands are listed in the Supplementary Materials (tables S1 to S10). Nanoswitches were constructed as described previously (18 (link), 21 (link)). A genomic ssDNA (New England Biolabs, M13mp18) was linearized using targeted cleavage with the Bts CI restriction enzyme. The linearized ssDNA was then mixed with a molar excess of an oligonucleotide mixture containing backbone oligos and detectors and annealed from 90° to 20°C at 1°C min⁻¹ in a T100 Thermal Cycler (Bio-Rad, USA). Following construction, the nanoswitches were purified using LC purification (23 (link)) to remove excess Oligonucleotides. The concentration of purified nanoswitches was determined by measuring A260 absorbance with a Thermo Fisher Scientific NanoDrop 2000.

To compare the endonuclease activities of wild-type and I38T PA-Nter in the presence of selected compounds (baloxavir acid, luteolin, orientin), we used a gel-based endonuclease inhibitory assay. The single-stranded DNA substrate M13mp18 (New England Biolabs) was cleaved in vitro by either protein. Each reaction (10 µL) contained 1 μM protein (GST-PA-Nter wild type/GST-PA-Nter I38T mutant) in digestion buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween20, 1 mM MnCl₂, 10 mM MgCl₂, 1 mM 2-mercaptoethanol) and was incubated with various concentrations of inhibitors (BXA/LU2/OTN/21/30). Reactions were initiated by the addition of 0.2 μg of M13mp18 plasmid. Reactions were incubated at 37 °C for 5 h and stopped by adding 1 μL of 0.2 M EDTA. Finally, cleavage of DNA substrate was visualized by agarose electrophoresis using 1% agarose gel stained with GelRed.

The in vitro NTPase activity of Twm1 was determined using a protocol adapted from Jemt, Farge [7 (link)]. NTP hydrolysis was measured by the release of phosphate using the malachite green phosphate assay. Reactions (20 μl) were prepared containing 20 mM Tris-HCl (pH 7.5), 4.5 mM MgCl₂, 1 mM DTT, 1 mM NTP and 25–100 ng of purified Twm1. Reactions were prepared in the presence or absence of DNA and incubated at 21 °C for 4 h. DNA templates used were linear 15 bp dsDNA (FHA0), circular ssDNA (M13mp18; New England Biolabs) and linear 15 nucleotide ssDNA (FHA 3.1). Following incubation, reactions were diluted 2.5-fold in milli-Q sdH₂O and dispensed into a 96 well microtiter plate, to which 100 μl of malachite green solution was added. The OD_620nm was measured on a CLARIOstar (BMG Labtech), and sample values were normalized based on values obtained for controls. The final concentration of released phosphate was determined from a standard curve of known phosphate concentrations. Malachite green solution and phosphate standards were prepared as described previously [62 (link)]. Experiments were repeated for each sample at least five times, with each experiment performed in triplicate. p values were calculated using the Student’s t-test function in Microsoft Excel.

Primer extension reactions were performed at 30°C in polymerisation buffer (25 mM Tris-HCl pH 7.5, 8 mM Magnesium Acetate, 5 mM Potassium Glutamate, 5% Glycerol). Purified proteins used in the primer extension assays were purified as previously described (Devbhandari and Remus, 2020 (link)). DNA template for the assay was generated by annealing a primer (5′-CCCAGTCACGACGTTGTAAAACG-3′) to M13mp18 single-stranded DNA (New England Biolabs, N4040S). The assay was initiated by incubation of 1 nM of DNA template with 1mM ATP, 1mM DTT, 80 μM dATP, 80 μM dGTP, 80 μM dCTP and 400 nM of RPA for 5 min. PCNA and RFC were then added to 70 nM and 4 nM, respectively, and incubation was continued for 5 min. Then, 33 nM of α−³²P-dATP (3000 Ci per mmol) and 4 nM of either Pol δ^WT or Pol δ^D941Y was added to the reaction, resulting in a primer extension by nine base pairs (due to lack of dTTP). After 5 min, 80 μM dTTP were added to the mix for synchronous primer extension. Equal volume aliquots of this reaction (18 μl) were removed from the master reaction (100 μl) at indicated times and stopped by adding EDTA and SDS to final concentrations of 40 mM and 0.25%, respectively. Products were fractionated on a 0.8% alkaline agarose gel (30 mM NaOH and 2 mM EDTA), dried and imaged using Typhoon FLA 7000. Quantification of the gel images was performed using ImageJ.