Typhoon 9500

EMSA was performed with DNA fragments (100 ng) mixed with 2 μM purified DR0997 or DR0615 protein in a total volume of 10 μl. The binding buffer contained 10 mM Tris-HCl (pH7.5), 10 mM KCl, 200 mM NaCl, 5 mM MgCl₂, 5 mM MnCl₂, 10 μg mL^–1 bovine serum albumin, 200 μM cAMP, and 100 μM dithiothreitol[44 (link)]. The reaction mixture was incubated at 30°C for 30 min and then loaded onto 12% (w/v) polyacrylamide gels in 1×Tris-borate-ethylenediamine tetraacetic acid buffer. Electrophoresis was performed at 100 V for 6 h at 4°C, and the gel was stained with ethidium bromide and photographed (Typhoon 9500, GE Healthcare). The E. coli CRP-binding site was used to identify predicted CRP-regulated genes in D. radiodurans. The promoter of dr1998 was used as a positive control of DR0615. The dr0167 promoter and coding region of dr0167 (dr0167C) were used as negative controls. All primers are listed in S1 File.

For primer extension, 5 μg of total RNA isolated from log phase cultures of M. smegmatis or M. tuberculosis was heated to 65°C, rapidly chilled, followed by the addition of primers (which anneals to the 5′end of topoI coding region) and incubation at 55°C, again chilled and extension mix (containing 10 mM dNTP mix and 200 units RevertAid Premium Reverse Transcriptase) was added followed by incubation at 50°C for 1 h. The products were separated in a 6% sequencing gel. The size of the products was deduced using a sequencing reaction. For sequencing, the pUC-T7A1 plasmid and pUC forward primer were used. Gels were scanned in a Typhoon 9500 (GE) phosphorimager.

Reactions were carried out in RX buffer (10 mM HEPES pH 7.5, 10% glycerol, 140 mM KCl and 1 mM EDTA). 200 nM Pol γ was pre-incubated with 500 nM 5′-³²P-26 nt DNA (M13-26-primer in Table 1) for 5 min at 37°C. Reactions were initiated by the addition of 10 mM MgCl₂, and quenched at the indicated times by addition of four reaction volumes of Q buffer (80% formamide, 1% SDS, 25 mM EDTA, pH 8.0) and heated at 95°C for 5 min. Reaction products were resolved by electrophoresis on a 20% denaturing (7M urea) polyacrylamide gel and quantified on a GE Typhoon 9500 at 100 μm using ImageQuant 7.1. The apparent rate of substrate digestion was estimated by fitting the time-dependent fraction of substrate reduction from the initial rate for untreated and H₂O₂ oxidized Pol γ.

Standard exonuclease assays were carried out in a 10 μL final volume containing 20 mM HEPES-KOH, pH 7.5, 50 mM KCl, 0.5 mM TCEP, 10 mM MgCl₂, 0.05% (v/v) Triton X-100, 5% (v/v) glycerol, and SNM1 proteins as indicated. Inhibitors were dissolved in DMSO and diluted immediately prior to the reaction in the above buffer. Inhibitors were serially diluted two-fold from 100 μM. The DMSO concentration was kept constant at 0.1% (v/v) in the final reaction mixture. Inhibitors were incubated with the indicated SNM1 enzyme for 10 minutes at room temperature; reactions were initiated by addition of 10 nM DNA substrate and incubated at 37 °C for 20 min. Reactions were quenched by addition of 5 μL stop solution (95% formamide, 10 mM EDTA, 0.25% (v/v) xylene cyanol, 0.25% (v/v) bromophenol blue) and heated at 95 °C for 3 min. Reactions were analysed by 20% denaturing polyacrylamide gel electrophoresis solution of 19 : 1 acrylamide:bis-acrylamide, BioRad and 7 M urea (Sigma Aldrich) in 1 × TBE (Tris-borate EDTA) buffer. Electrophoresis was carried out at 525 V for 75 minutes; gels were subsequently fixed for 60 minutes in a 50% (v/v) methanol, 10% (v/v) acetic acid solution, and dried at 80 °C for two hours under vacuum. Dried gels were exposed to a Kodak Phosphor imager screen and scanned using a Typhoon 9500 instrument (GE Healthcare), scanned images were quantified using ImageJ.

Standard nuclease assays were carried out in reactions containing 20 mM HEPES–KOH, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 0.05% (v/v) Triton X-100, 5% (v/v) glycerol (final volume: 10 μl), and the indicated concentrations of Artemis. Reactions were started by addition of substrate (10 nM), incubated at 37°C for the indicated time, then quenched by addition of 10 μl stop solution (95% formamide, 10 mM EDTA, 0.25% (v/v) xylene cyanole, 0.25% (v/v) bromophenol blue) with incubation at 95°C for 3 min.
Reaction products were analysed by 20% denaturing polyacrylamide gel electrophoresis (made from 40% solution of 19:1 acrylamide:bis-acrylamide, BioRad) and 7 M urea) in 1× TBE (Tris–borate–EDTA) buffer. Electrophoresis was carried out at 700 V for 75 min; gels were subsequently fixed for 40 min in a 50% methanol, 10% acetic acid solution, and dried at 80°C for 2 h under a vacuum. Dried gels were exposed to a Kodak phosphor imager screen and scanned using a Typhoon 9500 instrument (GE).