Boundaries of the pSC101 minimal replicon are conditional.
The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.
Miller
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CA
Department of Genetics, Stanford University School of Medicine, California 94305, USA.
Ingmer
H
H
Cohen
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SN
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D034982:Integration Host Factors; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011506:Proteins; D012045:Regulatory Sequences, Nucleic Acid; D018741:Replication Origin / Q000235:genetics; D012093:Replicon / Q000235:genetics*; D015183:Restriction Mapping; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D034982:Integration Host Factors; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I