Abstract
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.
Highlights
Cell viability requires the complete and precise duplication of the entire genome in a timely manner
This study has determined the stability of a replication fork in vivo that has stalled because of a nucleoprotein block formed by an array of tetracycline repressor-operator site complexes
This Fluorescent Repressor Operator System (FROS) system is able to cause a replisome to stall in a known location on the chromosome and the replication status of the array can be determined visually using fluorescence microscopy and verified with neutralneutral 2D agarose gels
Summary
Cell viability requires the complete and precise duplication of the entire genome in a timely manner. If a replication fork encounters any of these roadblocks, the replisome may disengage, at least partially, from the DNA, allowing processing of the DNA into a structure that facilitates reloading of the replication proteins and restart of replication. This process may allow access of DNA repair factors, accessory helicases and homologous recombination proteins which can repair or bypass the blocking lesions. The fate of the replication proteins when encountering such impediments is uncertain, the replisome is thought to remain stable for an extended period of time at protein roadblocks before it is removed from the DNA [4,5,6]. Evidence suggests replisomes that have stalled owing to head-on collisions with transcription complexes remain stable for 60 min or more [7]
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