Article
- The EMBO Journal (2006) 25, 2596 - 2604
- doi:10.1038/sj.emboj.7601155
Published online: 25 May 2006
Subject Categories:
Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo
Christophe Possoza, Sergio R Filipeab, Ian Grainge and David J Sherratt
- Division of Molecular Genetics, Department of Biochemistry, University of Oxford, Oxford, UK
Correspondence to:
David J Sherratt, Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 275 296; Fax +44 1865 275 297; E-mail: sherratt@bioch.ox.ac.uk
aThese authors contributed equally to this work
bPresent address: ITQB/UNL, Av. da Republica (EAN), Apartado 127, 2781-901 Oeiras, Portugal
Received 3 February 2006; Accepted 25 April 2006
Abstract
We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.
Keywords:
- Escherichia coli,
- recombination,
- replication fork stalling,
- repressor binding,
- restart
