Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA.
The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli
Health Canada, Biologics and Genetics Therapies Directorate, Center for Biologics Research, Banting Bldg, 2201C, Tunney's Pasture, Ross Avenue, Ont, K1A 0L2, Ottawa, Canada.
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Zoueva
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Iyer
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Kozlowski
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D012093:Replicon; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases