Incorporation of exogenous circular DNA into large catenated networks in isolated nuclei. Evidence for involvement of the nuclear scaffold.
Circular plasmid DNA was efficiently converted into huge catenated intranuclear networks by incubation with isolated nuclei in the presence of ATP. The network production is abolished by omission of ATP, strongly inhibited by etoposide (VP-16), but only slightly inhibited by antibody to topoisomerase I, indicating that the major enzyme responsible for catenation is DNA topoisomerase II. Under optimal conditions, a single nucleus incorporates about 4.2 x 10(4) DNA rings into its networks. Under the light microscope, networks retrieved from nuclei appear like spheres of various sizes. Sedimentation analysis showed that most of the networks are composed of thousands of catenated rings, which was confirmed by electron microscopy. Data from experiments that caused partial disruption of the networks were submitted to analysis based on probable models of catenane structure. The results suggest that the predominant pattern is a linear alignment of catenated rings. Similar networks are formed when the nuclear scaffold is incubated with circular DNA in the presence of nuclear extract containing topoisomerase II. Titration experiments showed that the scaffold binds a stoichiometric amount of the substrate and that a critical level of DNA is required for network formation. The results are consistent with the idea that DNA-binding sites are fixed on the scaffold and mediate catenation of bound DNA circles by holding them in close proximity to each other. We propose that catenation by the nuclear scaffold also occurs in intact nuclei, suggesting additional roles for the scaffold in vivo.