Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy.
We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times.
Kuzminov
A
A
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Schabtach
E
E
Stahl
F W
FW
D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D008854:Microscopy, Electron / Q000379:methods*; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D018741:Replication Origin
D004270:DNA, Circular; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D015245:Deoxyribonuclease BamHI; C052380:endodeoxyribonuclease NcoI; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific