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The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome
Author(s) -
Weigel Christoph,
Messer Walter,
Preiss Susanne,
Welzeck Michaela,
Boye Erik
Publication year - 2001
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2001.02409.x
Subject(s) - dnaa , biology , minichromosome , origin of replication , genetics , seqa protein domain , minichromosome maintenance , mutant , chromosome , dna replication , origin recognition complex , plasmid , mutation , circular bacterial chromosome , control of chromosome duplication , pre replication complex , microbiology and biotechnology , dna , eukaryotic dna replication , gene
We have developed a simple three‐step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild‐type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild‐type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild‐type cells. Mutations in the left half of oriC , in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC 14 and oriC 21, mutated oriC s could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriC s is far better on the chromosome than on a minichromosome.