Nebuffer 4

To measure the FI of DraIII and its mutants, a two-fold dilution series of the concentrated protein stock solution was made using Diluent B (300 mmol/L NaCl, 10 mmol/L Tris-Cl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 500 μg/mL BSA, 50% glycerol; NEB) to give 21 decreasing concentrations of the enzyme (1×, 0.5×, 0.25×, etc.). Two microliters of the diluted enzyme solutions were then mixed with 1 μg λ DNA in a reaction volume of 20 μL in NEBuffer 4 (50 mmol/L potassium acetate, 20 mmol/L Tris-acetate, 10 mmol/L magnesium acetate, 1 mmol/L dithiothreitol, pH 7.9 at 25°C; NEB). The reactions were incubated at 37°C for 1 h and were then quenched by 2 μL of Stop Solution (10×, 200 mmol/L EDTA, 100 mmol/L Tris-Cl, pH 8.0, 0.03% bromophenol blue, 0.94% SDS). The quenched reactions were analyzed by agarose gel electrophoresis. Gel images were obtained using a UV imager (Bio-Rad) on ethidium bromide (EB)-stained gels. The FI was calculated as the ratio of the highest enzyme dilution showing no star activity to the lowest dilution showing complete digestion (Wei et al., 2008 (link)). The FIs were determined with purified proteins except for T181Y. The FI of T181Y was determined using cell lysate dilutions.

Fin tissue samples from individuals selected for RAD genotyping were used for DNA extraction using QIAGEN DNeasy 96 kits. Quantification of extract DNA was done using Invitrogen Quant-It pico green reagent and a PerkinElmer Victor V fluorimeter. Of the 456 samples chosen for inclusion in RAD library preparation, 27 had insufficient DNA concentration after extraction and quantitation. DNA extracts from the remaining 429 samples were normalized to 5 ng/μL and 500 ng of each sample was digested with Sbf1-HF restriction enzyme in NEBuffer 4 (New England Biolabs). Barcoded adapters were then ligated onto the cut ends of the restriction sites using T4 DNA ligase (New England Biolabs) and the samples were then pooled into libraries of 48 individuals each. The remaining steps of library preparation were carried out as described in Miller et al. (2012) (link) and Hecht et al. (2012) (link). The concentration of a 1:1000 dilution of each completed library was determined by quantitative polymerase chain reaction using Life Technologies PowerSYBR reagent and Kappa biosystems Illumina library DNA standards run on an Applied Biosystems 7900 instrument. Library concentration ranged from 6.5 to 71 nM after the addition of the P2 adapter and 15 cycles of PCR amplification. The concentration of each library was normalized to 5nM and sequenced on an Illumina HiSeq 2000 instrument.

The AP sites were cleaved by resuspending the bead-DNA complexes in 20 µl reaction solution containing 1× NEBuffer 4 and 10 U APE1 (New England BioLabs, M0282) with incubation at 37 °C for 2 h. Incubation without APE1 enzyme was performed to serve as an untreated control. After collecting the beads with a magnetic stand, the supernatant was collected and purified with 2× volumes of VAHTS DNA Clean Beads and eluted in 15 µl H₂O.

Human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), human apurinic/apyrimidinic endonuclease 1 (APE1), 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl₂, 10 mM DTT, pH 7.9), 10× ThermoPol reaction buffer (200 mM Tris–HCl, 100 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO₄, 1% Triton X-100, pH 8.8) and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris–acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs Inc. (Beverly, MA, U.S.A.). All HPLC-purified oligonucleotides (Table 1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

The E. coli Topo I WT and mutant proteins were expressed and purified as described in previous study53 (link). Commercial product of E. coli Topo I (NEB) was also tested and its RNA Topo activity was found to be slightly higher than that of the in-house prepared WT enzyme, probably due to the contamination of E. coli Topo III (Supplementary Fig. 8). The reaction buffer contained 1 × NEBuffer 4 (NEB, 1 × buffer: 50 mM KOAc, 20 mM Tris-acetate, 10 mM Mg(OAc)₂, 1 mM DTT, pH 7.9 at 25 °C) and 100 μg ml⁻¹ BSA. The concentration of substrates and proteins were described in the text. Reactions were quenched by phenol–chloroform extraction followed by ethanol precipitation. The reactions were then analysed by dPAGE.