Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones

Abstract

Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA.