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. 2009 Apr 3;284(14):9341–9353. doi: 10.1074/jbc.M900023200

FIGURE 8.

FIGURE 8.

The binding of cc-dsDNA to Mhr1 and the topological status of Mhr1-bound cc-dsDNA. A, the binding of cc-dsDNA to Mhr1. Negatively supercoiled pUC18 plasmid DNA (41.8 μm) was mixed with the indicated concentrations of Mhr1 in standard buffer and incubated at 37 °C for 30 min. The reaction mixtures containing the Mhr1-cc-dsDNA complexes were then subjected to a gel shift assay, as previously described (19). B, effects of increasing concentrations of Mhr1 on relaxed cc-dsDNA in the presence of calf thymus topoisomerase I (Top I). Relaxed cc-dsDNA was prepared by incubating negatively supercoiled pUC18 plasmid DNA (41.8 μm) with 1.0 unit/μl calf thymus topoisomerase I at 37 °C for 30 min. The relaxed cc-dsDNA (41.8 μm) was mixed with the indicated concentrations of Mhr1 in standard buffer and incubated in the presence of 0.2 units/μl topoisomerase I at 37 °C for 30 min. As a control, negatively supercoiled cc-dsDNA was incubated with the indicated concentrations of Mhr1 under the same conditions, without topoisomerase. After a phenol/chloroform extraction, the DNA products were analyzed by electrophoresis on 1% agarose gels for 11 h at room temperature.