TOPOISOMERASES: INVOLVEMENT IN DNA SYNTHESIS IN PROKARYOTES AND EUKARYOTES.

Abstract

The involvement of topoisomerase in DNA replication in a prokaryotic and a eukaryotic system was examined. The mechanism of in vitro DNA replication by an isolated replicative enzyme complex was also investigated. In bacteriophage T4 there is evidence that the type II topoisomerase coded for by the phage is involved in the initiation of replicative growing points. We have looked at the topological structure of the replicating T4 nucleoid by sedimentation of the DNA in neutral sucrose gradients containing various amounts of ethidium bromide. It was determined that at no time after infection does the replicating T4 DNA contain any large amount of negative superhelicity. The absence of the phage topoisomerase did not affect the topology of the nucleoid. It was therefore concluded that the role of the T4 topoisomerase in initiating DNA synthesis in T4 was not exerted at the level of the general topology of the replicating T4 DNA. An isolation procedure for the T4 topoisomerase for pursuance of further studies was also described. New mammalian topoisomerases were shown to be stimulated by epidermal growth factor (EGF) in two cultured fibroblast cell lines. Topoisomerase activity was seen first in the cell cytoplasm and subsequently in the nucleus. The peak of topoisomerase activity in the nucleus corresponded to the peak of EGF-induced DNA synthesis in the cells. At least a part of the topoisomerase activity stimulated by EGF was shown to be due to a type II topoisomerase by the ATP-dependence of the activity. The topoisomerase activity in the cytoplasm was shown to exist in a non-soluble, sedimentable form. Further characterization of the topoisomerase containing complex isolated from the cytoplasm was carried out. The complex was seen to be non-membrane bound and complex. DNA polymerase and nucleoside diphosphate kinase activities were also demonstrated to be contained within the complex. It was shown that this cytoplasmic complex was capable of in vitro DNA replication. Many parameters of the in vitro DNA replication reaction were examined. The process was seen to mimic in vivo replication in several ways. The complex was shown to not only be able to elongate DNA but to initiate replication through the creation of a replication bubble.