FIGURE 1.

Assembly of β clamp in the presence of γ complex onto supercoiled or nicked DNA. A, 3′ nicked G-T heteroduplex (lanes 1 and 2) or 3′ G-T supercoiled heteroduplex (lanes 3 and 4) were incubated with ³²P-labeled β clamp and γ complex in the absence (lanes 1 and 3) or presence of 30 mm NaCl (lanes 2 and 4), β·DNA complexes cross-linked, and the products were resolved by electrophoresis through 1% agarose (“Experimental Procedures”). DNA was visualized after ethidium staining (left panel) and β·DNA complexes scored by phosphorimaging (right panel). The positions of the nicked open circular (oc) DNA, supercoiled (sc) DNA, and free β are shown on the right. The stoichiometry of β loading was determined from percentage of the total signal in each lane. B, lifetime of β·DNA complexes on supercoiled G-T heteroduplex DNA was determined by loading [³²P]β in the absence of NaCl, followed by the addition of NaCl to 100 mm to prevent further loading. Incubation was continued, and the samples were removed and scored for β·DNA complexes (“Experimental Procedures”). Lanes 1–5, samples removed 0, 5, 10, 20, and 30 min after 100 mm NaCl addition. Lanes 6–10, assembly reaction was performed in the presence of 100 mm NaCl, and the samples were taken at 0, 5, 10, 20, and 30 min. β·DNA complexes were visualized as in A.