Stability of blocked replication forks in vivo.
Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein 'roadblocks' and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated.
School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
Karla A
KA
Mettrick
School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia ian.grainge@newcastle.edu.au.
Ian
I
Grainge
D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053488:DnaB Helicases / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D048168:Escherichia coli K12 / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D009154:Mutation; D013696:Temperature
D004269:DNA, Bacterial; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; C506730:dnaB protein, E coli; D053488:DnaB Helicases