Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro

Abstract

oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated by DNA gyrase alone in the absence of salt. Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt. Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt. A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis. Topoisomerase I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system. The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.