title: Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.
abstract: Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.
authors.affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom.
authors.forename: Milind K
authors.initials: MK
authors.lastname: Gupta
authors.forename: Colin P
authors.initials: CP
authors.lastname: Guy
authors.forename: Joseph T P
authors.initials: JT
authors.lastname: Yeeles
authors.forename: John
authors.initials: J
authors.lastname: Atkinson
authors.forename: Hazel
authors.initials: H
authors.lastname: Bell
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lloyd
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Marians
authors.forename: Peter
authors.initials: P
authors.lastname: McGlynn
mesh_terms: D016923:Cell Death; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism; D011485:Protein Binding; D014158:Transcription, Genetic; D000099272:Replisomes
chemical_list: D004265:DNA Helicases; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009698:Nucleoproteins; D000099272:Replisomes; C506744:rep protein, E coli