Expression and labelling of T7 endonuclease I

The variants of endonuclease I used in this study were expressed from the plasmid pET-TEV-endoI⁵¹ engineered to remove the endogenous cysteine (C113S mutation) and add a new one at an accessible position remote from the DNA interacting surface (S29C mutation) for labelling. These mutations were shown previously not to affect significantly the activity of the expressed protein⁴¹. For simplicity, we refer to this version of endonuclease I as wild type. The active site mutant carried the additional D55A mutation, while the Δ16 mutant had 16 residues truncated from the N-terminus³⁹.

BL21 (DE3) pLysS cells, transformed with the relevant plasmid, were grown at 37 °C to an A₆₀₀ of 0.6 in LB medium supplemented with carbenicillin and chloramphenicol. They were then cooled to 25 °C, supplemented with 0.1 mM IPTG to induce protein expression and grown at 25 °C for a further 4 h. Cells were harvested by centrifugation at 8000 g for 30 min, washed with PS buffer (50 mM NaH₂PO₄/Na₂HPO₄ (pH 8), 1 M NaCl) and stored at −80 °C.

Cells pellets were thawed on ice, resuspended in 5 vol (w/v) of lysis buffer (PS supplemented with 20 mM imidazole, 0.2% Triton X100, cOmplete (EDTA-free) protease inhibitor cocktail (Roche) and 1 mg/ml lysozyme) and incubated at 20 °C for 30 min. The lysate was sonicated, clarified by centrifugation at 20,000 g, 4 °C for 30 min and applied to a His-Trap FF column (Cytiva) equilibrated in PS + 20 mM imidazole. His-tagged endonuclease I was eluted with a linear gradient from 20 mM to 1 M imidazole in PS buffer and dialysed into 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM DTT. The N-terminal oligohistidine tag was cleaved off by addition of His-tagged TEV protease to a molar ratio of 1:50 and incubation at 16 °C for 16 h. Finally, endonuclease I was purified on a His-Trap FF column as described above, dialysed into endonuclease I storage buffer (50 mM Tris-HCl pH 7.5, 40 mM NaCl, 30% glycerol) and stored at −80 °C.

Prior to labelling, endonuclease I was thawed on ice and incubated with 20 mM DTT on ice for 1 h to reduce its cysteine residues fully. The DTT was then removed by exchanging the buffer to labelling buffer (50 mM NaH₂PO₄/Na₂HPO₄, pH 7.2, 100 mM NaCl) using a HiTrap Desalting column (Cytiva). This was used to resuspend a 3- to 5-fold molar excess of Cy3 (or Cy5) maleimide (GE Healthcare) and the reaction mix was incubated at 20 °C for 16 h. The reaction was quenched by the addition of 1 mM DTT and the majority of the unincorporated dye was removed by buffer exchange into 20 mM NaH₂PO₄/Na₂HPO₄ (pH 6.0), 40 mM NaCl. The labelled protein was further purified on a HiTrap SP HP column (Cytiva) and eluted with a linear 40 mM – 2 M NaCl gradient in the same buffer. Finally, the protein was dialysed into endonuclease I storage buffer and stored at −80 °C. The purity of the endonuclease I variants used in this study is shown in (Supplementary Fig. ^2A–C).