Figure 9.

HMGB1 enhances ATP hydrolysis by topoisomerase IIα. (A) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. (B) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ-³²P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO₄) was measured by thin layer chromatography (24) and quantified by PhosporImaging. Data represent the average of two independent experiments.