Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus

Abstract

The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.