Nucleic Acids Research, 1993, Vol. 21, No. 8 1689-1695
© 1993
MOLECULAR BIOLOGY |
Topoisomerase activity associated with SV40 large tumor antigen
Département de Microbiologie, Faculté de Médicine, Université de Sherbrooke Sherbrooke, Québec J1H 5N4, Canada 1School of Life and Health Sciences, University of Delaware Newark, DE 19716, USA
*To whom correspondence should be addressed
Received March 3, 1993. Accepted March 3, 1993.
Purified preparations of simian virus 40 (SV40) large tumor antigen (LT) from three different sources, including LT expressed from a recombinant baculo-virus, were found to relax negatively supercolled cyclic DNA molecules, whether or not they contained SV40 sequences. Relaxation was stimulated by MgCl2 but not by ATP, and Inhibited by camptothecin, suggesting the involvement of an enzymatic activity similar to that of topoisomerase I (topo I). However, the pH requirements for relaxation by respectively LT and topo I are different. Also, antibodies reacting with LT Inhibited relaxation by preparations of LT but not topo I, whereas antibodies inhibiting relaxation by topo I had no effect on relaxation by LT. Reconstruction experiments suggested that both procedures used to purify LT, immunoaffinity chromatography and DEAE-Sepharose chromatography, separate topo I from LT. Finally, relaxing activity was found in over 40 preparations of LT, and in the few instances where activity could not be found, it probably had been lost during storage, rather than absent from the start. Whereas these results seem to exclude that the activity being detected Is that of a contaminant of LT, they would be consistent with this activity being that of a stable topo-LT complex, or else intrinsic to LT itself.