FIG 3.

The DnaX complex stimulates Topo III-catalyzed unlinking of catenanes and precatenanes. Assays were performed as described in Materials and Methods. Reaction products were analyzed by electrophoresis through 1% agarose gels at 1.5 V/cm for 16 h at room temperature using 50 mM Tris-HCl (pH 7.8 at 23°C), 40 mM sodium acetate (NaOAc), and 1 mM EDTA as the electrophoresis buffer. Gels were dried and autoradiographed. Reaction products were quantified using Fuji MacBas software. (A) Topo IV-catalyzed unlinking of catenated DNA dimers (left panel) and precatenated DNA (right panel). (B) Topo III-catalyzed unlinking of catenated DNA dimers (left panel) and precatenated DNA (right panel). (C) The DnaX complex stimulates Topo III-catalyzed unlinking of catenated DNA dimers. The indicated proteins were present at 40 nM. Topo III was present at 1.2 nM. (D) The DnaX complex stimulates Topo III-catalyzed unlinking of precatenated DNA. The indicated proteins were present at 40 nM. Topo III was present at 1.2 nM. (E) Quantification of the amount of final product (nicked, form II [FII] DNA) from panel C (mean and standard deviation from three experiments). (F) Quantification of the amount of final product (late replication intermediate [LRI]) from panel D (mean and standard deviation from three experiments).