Fig 5.
Isolation of the ssDNA-MobMN243 stable complexes after IR1+12pA oligonucleotide cleavage. (A) Scheme of the substrate and reaction products expected after IR1+12pA nicking by MobMN243. (B) DNA analysis. IR1+12pA 5′-labeled (Cy5) oligonucleotide (60 nM) was incubated with different concentrations of MobMN243 (128, 256, and 512 μM) for 20 min at 30°C in the presence of 8 mM MnCl2. The reaction was stopped by incubation with 0.1% SDS and proteinase K (100 μg/ml) for 30 min at 37°C. Samples were loaded in a denaturing 20% PAA gel (7 M urea), and DNA was visualized in a phosphorimager platform. (C) Protein analysis. MobMN243 (10 μM) was incubated with DNA (10 or 20 μM) in the same conditions used for the DNA analysis. Samples were visualized by SDS-PAGE (12%) and SYPRO Ruby staining. A stable adduct corresponding to the MobMN243 protein attached to a 21-mer ssDNA fragment was identified (MobMN243-DNA21).