RECONSTITUTION OF PBR322 DNA REPLICATION IN VITRO WITH PURIFIED PROTEINS

Abstract

The replication of a duplex, covalently-closed supercoiled (form I) DNA as an intact molecule requires that the topology of the DNA be modulated. The role of proteins that alter DNA structure, e.g., topoisomerases and helicases, in this process is poorly understood. In order to understand the role of these activities during the replication of a form I DNA, a system composed of purified proteins capable of replicating pBR322 DNA has been developed.;The following proteins, which were expected to be required for pBR322 DNA replication, were purified; RNA polymerase holoenzyme, DNA polymerase I, ribonuclease H, DNA gyrase, topoisomerase I, DNA ligase, SSB, DNA polymerase III*, the dnaN, dnaB, dnaC, and dnaG gene products, replication proteins i, n, and n'', and factor Y (this collection of protein will be referred to as the complete system). In addition, the uvrD (helicase II) gene product was also purified.;The complete system of proteins supported extensive DNA synthesis and utilized only plasmid DNAs that contained ColE1-type origins of DNA synthesis as templates, such as pBR322 DNA. It was demonstrated that low levels of Topoisomerase I (Topo I) was required to generate the specificity inherent in the different mechanisms of H and L strand DNA synthesis as well as to discriminate between pBR322 and (SLASHCIRC)X174 as template DNAs.;Little of the synthetic products generated in a typical reaction, however, were form I DNA. An analysis of the DNA products formed at levels of Topo I sufficient for template discrimination demonstrated that the three major species were: multiply-interlinked catenated form I:form I DNA dimers; a discrete population of almost completely replicated DNA molecules; and long multigenome linear duplex DNA molecules. Thirty to fifty-fold higher levels of Topo I was required to catalyze the segregation of the daughter pBR322 DNA molecules. Genuine, form I DNA product matured from a last cairns-type replication intermediate through form II DNA and not through the form I:I DNA dimers. Evidence in support of this model is presented.