The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome
| Autor: | Erik Boye, Michaela Welzeck, Morigen, Christoph Weigel, Susanne Preiss, Walter Messer |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
DNA Replication
Mutant Replication Origin Biology Polymerase Chain Reaction Microbiology Plasmid SeqA protein domain Minichromosome Minichromosome maintenance Transduction Genetic Escherichia coli Molecular Biology Recombination Genetic Genetics fungi DNA replication Chromosome Sequence Analysis DNA Chromosomes Bacterial Flow Cytometry DnaA Blotting Southern Mutation bacteria Plasmids |
| Zdroj: | Molecular Microbiology. 40:498-507 |
| ISSN: | 1365-2958 0950-382X |
| DOI: | 10.1046/j.1365-2958.2001.02409.x |
| Popis: | 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 oriC14 and oriC21, mutated oriCs 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 oriCs is far better on the chromosome than on a minichromosome. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text