Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.
All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.
Payne
Bryony T I
BT
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
van Knippenberg
Ingeborg C
IC
Bell
Hazel
H
Filipe
Sergio R
SR
Sherratt
David J
DJ
McGlynn
Peter
P
D001426:Bacterial Proteins / Q000378:metabolism*; D004261:DNA Replication; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D056968:Lac Repressors; D009097:Multienzyme Complexes / Q000378:metabolism; D009875:Operator Regions, Genetic; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D000099272:Replisomes
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009097:Multienzyme Complexes; D012097:Repressor Proteins; D000099272:Replisomes; C539064:LacI protein, E coli