Fig. 6. ATP-dependent protein–protein interactions of BsSMC on DNA. (A) BMH cross-linking. Three different BsSMC proteins [wild type (lanes 1–6), K37I (lanes 7–12), S1090R (lanes 13–18); 450 nM arms] were mixed with the indicated nucleotides in the absence or presence of φX174 virion DNA (15.6 μM nucleotides). After incubation, the proteins were cross-linked with BMH, fractionated in a 2.5–7.5% SDS polyacrylamide gel, and analyzed by immunoblotting with an anti-BsSMC antibody. The positions of the intramolecular cross-linking products (asterisks) and the intermolecular cross-linking products (ATP-stimulated products, filled circles; ATPγS-specific products, open triangles) are indicated. The position of non-cross-linked BsSMC polypeptide (135 kDa) is shown in lane 0. The concentrations of KCl and MgCl₂ were 7.5 and 2.5 mM, respectively. (B) Spin-down assay. The same reactions were set up as in (A). After incubation, the mixtures were spun at 16 000 g for 15 min. The supernatants (upper) and pellets (lower) were separated, fractionated by 7.5% SDS–PAGE and stained with silver.