Replication of Colicin E1 Plasmid DNA in Cell Extracts

Cell extracts were prepared from Escherichia coli carrying colicin El plasmid. The DNA in extracts was almost exclusively closed-circular DNA of the plasmid. Labeled deoxyribonucleotides were incorporated into DNA in extracts. DNA of colicin E1 plasmid was the sole DNA product, and was composed of c...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1974-03, Vol.71 (3), p.802-806
Main Authors: Sakakibara, Yoshimasa, Tomizawa, Jun-Ichi
Format: Article
Language:English
Subjects:
Biochemistry
Biological Sciences: Biochemistry
Cell extracts
Cell-Free System
Centrifugation
Centrifugation, Density Gradient
Colicins - biosynthesis
DNA
DNA Replication - drug effects
DNA, Bacterial - analysis
DNA, Bacterial - biosynthesis
DNA, Circular - analysis
DNA, Circular - biosynthesis
Escherichia coli - metabolism
Extrachromosomal Inheritance
Hybridity
Kinetics
Molecular structure
Molecules
Nalidixic Acid - pharmacology
Phosphorus Radioisotopes
Plasmids
Product labeling
Ribonucleotides - metabolism
Rifampin - pharmacology
RNA
Thymine Nucleotides - metabolism
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Summary:Cell extracts were prepared from Escherichia coli carrying colicin El plasmid. The DNA in extracts was almost exclusively closed-circular DNA of the plasmid. Labeled deoxyribonucleotides were incorporated into DNA in extracts. DNA of colicin E1 plasmid was the sole DNA product, and was composed of completely replicated molecules and a class of replicative intermediates. The intermediates carried an average of approximately two pieces of DNA fragments that had a sedimentation coefficient of approximately 6 S and were not covalently attached to the parental DNA strands. The replication was initiated on closed-circular molecules and the complete molecules were synthesized semiconservatively. The DNA synthesis depended on the four ribonucleoside triphosphates and was sensitive to rifampicin. A round of DNA replication, once initiated, was completed in the presence of rifampicin, indicating that RNA synthesis is involved in the initiation of replication of the plasmid DNA. Most of the replicated molecules were isolated in super-coiled structures. These results indicate that this soluble system is capable of carrying out a complete round of replication of colicin El plasmid DNA.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.71.3.802