Figure 7.

Ability of β⁺, β^C and β⁺/β^C to be loaded on DNA. Loading of β^C (A), β⁺ (B) or β⁺/β^C (C) onto nicked DNA by DnaX 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 (B). An aliquot of the clamp loading reaction prior to gel filtration chromatography (load), as well as representative gel filtration fractions we analyzed by native agarose gel electrophoresis. Positions of free clamp (free β), and clamp loaded onto nicked DNA (β on DNA) are shown. Phosphorimager analysis displaying positions of radiolabeled clamp proteins is shown at the top of each panel, while results of ethidium bromide staining highlighting positions of nicked and supercoiled DNAs are shown at the bottom of each panel. Results from one representative experiment are shown. This experiment was repeated at least three times, and the amount of clamp loaded onto DNA or free in each experiment was determined using the Molecular Analyst software (BioRad).