Cyclone plus storage phosphor system

The DNA helicase activity was carried out as previously described with slight modification [73 (link)]. Briefly, the reaction was performed in a 20-μl mixture containing 20 mM Tris–HCl (pH 8.0) 0.1 mg/ml BSA, 1 mM DTT, 10 mM ATP, 5 mM MgCl₂, 5 nM DNA substrate, and the indicated amount of SisPINA at 55 °C for 30 min and stopped by the addition of 10 μl stop buffer (20 mM EDTA, 1%SDS, 2 mg/ml proteinase K [Takara], and 0.02% bromophenol blue). The samples were further incubated at 55 °C for 10 min. The samples (20 μl) were loaded onto an 8% native polyacrylamide gel. Electrophoresis was run at 130 V for 90 min in 1× TBE buffer [89 mM Tris-borate (pH 8.3) and 1 mM EDTA]. The gels were exposed to a phosphorimager. The phosphorimager was then scanned with the Cyclone Plus Storage Phosphor System (PerkinElmer).

The HJ cleavage assay was performed in a 20-μl mixture containing 25 mM Tris–HCl (pH 8.0) 50 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT, 2 mM ATP, 5 mM MgCl₂, JY HJ DNA or HSL HJ DNA, SisHjc, and the indicated amount of SisPINA. The reaction was incubated at 55 °C for 30 min unless otherwise indicated. After 30 min, the reaction products were analyzed by native or denaturing gel electrophoresis. For native gel electrophoresis, 10-μl stop buffer (same as for helicase assay) was added and incubated at 55 °C for another 10 min, and then, 20-μl samples were loaded onto an 8% native gel. For denaturing gel electrophoresis, 2 μl proteinase K (20 mg/ml) and 2 μl 10% SDS were added and incubated at 55 °C for 5 min. Then, 20-μl denaturing buffer (95% formamide, 10 mM EDTA, and 0.02% bromophenol blue) was added and incubated at 95 °C for another 5 min. The products were immediately transferred to 4 °C for 5 min. Reaction mixture (30 μl) was loaded onto a 15% denaturing polyacrylamide gel containing 8 M urea. The gels were exposed to a phosphorimager. The phosphorimager was then scanned with the Cyclone Plus Storage Phosphor System (PerkinElmer).