DECATENATING ACTIVITY OF ESCHERICHIA-COLI DNA GYRASE AND TOPOISOMERASE-I AND TOPOISOMERASE-III DURING ORIC AND PBR322 DNA-REPLICATION IN-VITRO

oriC and pBR322 DNA replication, reconstituted with purified replicati on proteins, has been used to study the functional activities of Esche richia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the oriC system, DNA gyrase-ca talyzed decatenation of daughter DNA molecules was very inefficient, w hereas topoisomerase III could catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules c ould be decatenated by DNA gyrase alone in the absence of salt. Decate nation by DNA gyrase in the pBR322 system was completely inhibited, wi thout a concomitant inhibition of DNA synthesis, by the addition of ph ysiological concentrations of salt. Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, eve n in the presence of high concentrations of salt. A similar effect cou ld not be observed in the oriC system, because the addition of salt in hibited DNA synthesis. Topoisomerase I was incapable of catalyzing dec atenation under any conditions examined in either the oriC or pBR322 r eplication system. The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.