DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes

Abstract

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.

Unwinding of chromatin by the SV40 large T antigen DNA helicase.

“…Our results confirm previous observations, showing that topoisomerase cleavage sites are more or less repressed in DNA covered by nucleosomes (Capranico et al, 1990;Richter and Ruff, 1991) and imply that specific mechanisms are essential for topoisomerases to efficiently act on chromatin during replication. Accordingly, topoisomerase I, which appears to be positioned near the forks of replicating SV40 minichromosomes in vivo (Avemann et al, 1988;Hamelin et al, 1994; for a review see also Richter and Knippers, 1989), possibly must associate with the replication apparatus to obtain adequate access to parental strands. Formation of such a functional complex (see also Mann, 1993;Marton et al, 1993) may require several (replication) factors, because neither DNA polymerase ax nor replication protein A could support the unwinding of ccc chromatin in our hands (U.Ramsperger and H. Stahl, unpublished results).…”

“…This implies that topoisomerases do not have sufficient access to nucleosomally organized DNA to relax in time the numerous positive superhelical turns originating from unwinding of ccc chromatin. The native minichromosomes used contained bound topoisomerases from in vivo sources (see also Hamelin and Yaniv, 1979;Hamelin et al, 1994), and extensive unwinding by T antigen was also not observed with ccc chromatin that had been assembled in vitro in the additional presence of human topoisomerase I (data not shown). Therefore, turnover rates of topoisomerases appeared to be inhibited, even if they were allowed to bind the DNA before chromatin assembly.…”