Figure 8.

Ability of β⁺, β^C and β⁺/β^C to be unloaded from DNA by DnaX complex. The ability of DnaX to unload β⁺ (A, B) or β⁺/β^C (C, D) from DNA was analyzed as described in ‘Materials and Methods’ section. The myc-tagged β⁺/his₆-tagged β⁺ protein (β⁺/β⁺) was used for the β⁺ results shown in (A) and (B). Positions of free clamp (free β), and clamp loaded onto nicked DNA (β on DNA) are shown. Results of both phosphorimager analysis, illustrating positions of radiolabeled clamp proteins (A, C), as well as ethidium bromide staining, highlighting positions of nicked circular, linear and supercoiled DNAs (B, D) are shown. DnaX was used at the following concentrations: lane 1, 0 nM; lane 2, 10 nM; lane 3, 100 nM; lane 4, 1 μM; lane 5, 2.5 μM; lanes 6 and 7, 5 μM. We were unable to reliably quantitate the level of β⁺/β^C clamp unloading catalyzed by DnaX, due to the poor signal-to-noise ratio.