Initiation of unidirectional ColE2 DNA replication by a unique priming mechanism.
The ColE2 DNA can be replicated in an in vitro system consisting of a crude extract of Escherichia coli cells. DNA synthesis requires a plasmid-coded protein (Rep) and host DNA polymerase I but not host RNA polymerase. Replication starts at a fixed region containing the origin and proceeds unidirectionally. The leading- and lagging-strand DNA fragments synthesized around the origin were identified from early replicative intermediates. The 5' end of the leading-strand DNA fragment was mapped at a unique position in the minimal origin and carried RNA of a few residues. The results suggested that the initiation of the leading-strand DNA synthesis does not require the host DnaG protein. Thus the Rep protein itself seems to be a primase. Synthesis of the primer RNA at a fixed site in the origin region on a double-stranded DNA template is a unique property of the ColE2 Rep protein among other known primases. The 3' end of the lagging-strand DNA fragment was mapped at a unique position just at the end of the minimal origin region. Termination of the lagging-strand DNA fragment at that position seems to be the mechanism of the unidirectional replication of ColE2 plasmid.
D001426:Bacterial Proteins / Q000378:metabolism; D003086:Bacteriocin Plasmids / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases; D019915:DNA Primase; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000235:genetics*; D012316:RNA Nucleotidyltransferases / Q000502:physiology; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D010448:Peptide Initiation Factors; C034477:RNA primers; D012329:RNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases