Figure 5.

TopoI catalysed catenation and decatenation. (A) UCSC browser view for Ter-region in higher magnification indicating the specific recruitment (FC) and action peaks (D108A and IT) for TopoI. (B) Primer extension of genomic DNA to confirm D108A induced cleavage at Ter region. Genomic DNA was isolated from MsTopoI D108A overexpressing M. smegmatis cells. Primer extension was carried out using forward primer (Ms_3434_3432_F) with Taq DNA polymerase and analysed as described in the Materials and Methods and legends for figure 5. (C) Catenation assay with single-stranded M13 DNA. 0.2 μM of TopoI was incubated with 100 ng of M13 ssDNA in a buffer containing 20 mM Tris (pH 8.0), 20 mM KCl, 6 mM MgCl₂, 5 mM spermidine, 50 μg/ml BSA and 0.5 mM DTT in the presence of 30% glycerol or 10% PEG-400 or 10% PEG-8000. The formation of catenated products were analysed on 0.8% agarose gels. (D) Decatenation assay with kDNA. 0.2 uM of TopoI was incubated with 200 ng of kDNA in a buffer containing 20 mM Tris, 20 mM NaCl, 5 mM MgCl₂. The mini circles released by the action of TopoI were analysed on 0.7% agarose gels. Control: reaction mixture without TopoI.