Figure 9.

MukB hinge domain fragment binds DNA. A, wild-type, but not KE,RE, MukB hinge domain induces supercoils in the nicked DNA substrate. Either wild-type or KE,RE hinge domain fragment was incubated with the nicked DNA substrate for 5 min at 37 °C, NAD and E. coli DNA ligase were then added, and the products were analyzed as in the legend to Fig. 1B. B, densitometric lane traces of the gel shown in A. C, wild-type, but not KE,RE, hinge domain fragment cross-links to DNA. The indicated concentrations of wild-type, KE,RE hinge domain fragment, or full-length MukB were incubated with a 5′-³²P-labeled 100-mer DNA duplex for 5 min at 37 °C. Formaldehyde was then added to 0.7%, and the incubation continued for 30 min on ice. The HCHO was then neutralized by the addition of Tris base, and the DNA-protein complexes were analyzed by electrophoresis through 4–12% gradient SDS-PAGE. One percent SDS was added to the reactions shown in lanes 3, 5, and 7 prior to the addition of HCHO.