DNA STRAND PASSING AND THE FUNCTION OF TYPE II DNA TOPOISOMERASES

Abstract

ABSTRACT A DNA topoisomerase from bacteriophage T4-infected E. coli cells has recently been isolated and characterized. The T4 DNA topoisomerase catalyzes the relaxation of superhelical DNAs (whether positively or negatively coiled) in a reaction requiring ATP hydrolysis. The purified enzyme contains multiple subunits, which are apparently coded for by T4 genes 39, 52 and 60. Genetic and biochemical studies of mutants in these genes indicate that the T4 DNA topoisomerase is essential for phage T4 DNA replication and is most likely involved in the initiation of DNA replication forks. Mechanistic studies of this enzyme have clearly established that T4 DNA topoisomerase (as well as other type II DNA topo-isomerases) catalyzes the so-called “DNA strand passing reaction,”presumably via mechanisms that involve a transient double-chain scission on one of the two crossing DNA double helices. The passage of a second double-stranded DNA segment through this transient double-strand break results in a variety of DNA topoisomerization reactions, including knotting:unknotting; relaxation: supercoiling and catenation:decatenation. Using various assays specific for the type II DNA topoisomerases (such as unknotting, catenation and decatenation), an ATP dependent enzyme activity has been detected in all eukaryotic organisms tested thus far and highly purified from HeLa cells and calf thymus. The highly purified eukaryotic type II DNA topoisomerase (designated as topo II) is strikingly similar to the T4 DNA topoisomerase. The possible roles of such type II enzymes in a variety of biological functions will be discussed. A new eukaryotic type I DNA topoisomerase (designated as topo I) has also been purified to near homogeneity from HeLa cells. This type I DNA topoisomerase is a major nuclear protein and is most likely ubiquitous. Its possible relationship to the eukaryotic “nicking-closing” enzyme is discussed.