Replication fork reversal occurs spontaneously after digestion but is constrained in supercoiled domains.
Replication fork reversal was investigated in undigested and linearized replication intermediates of bacterial DNA plasmids containing a stalled fork. Two-dimensional agarose gel electrophoresis, a branch migration and extrusion assay, electron microscopy, and DNA-psoralen cross-linking were used to show that extensive replication fork reversal and extrusion of the nascent-nascent duplex occurs spontaneously after DNA nicking and restriction enzyme digestion but that fork retreat is severely limited in covalently closed supercoiled domains.
Fierro-Fernández
Marta
M
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Hernández
Pablo
P
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Krimer
Dora B
DB
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Schvartzman
Jorge B
JB
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address: schvartzman@cib.csic.es.
D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D053904:DNA Breaks, Single-Stranded; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry*; D004278:DNA, Superhelical; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005363:Ficusin / Q000737:chemistry; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D005363:Ficusin