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Nucleic Acids Research 2005 33(21):6931-6941; doi:10.1093/nar/gki994
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Published online 6 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Kathrin Marheineke*, Olivier Hyrien and Torsten Krude1,*

Laboratoire de Génétique Moléculaire, UMR CNRS 8541 Ecole Normale Supérieure, 46 rue d'Ulm, 75 230 Paris Cedex 05, France 1Department of Zoology, University of Cambridge Downing Street, Cambridge CB2 3EJ, UK

*To whom correspondence should be addressed. Tel: 0033 1 44 323733; Fax: 0033 1 44 323941; Email: marheine{at}wotan.ens.fr

Received September 13, 2005. Revised November 3, 2005. Accepted November 15, 2005.

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G1 phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.

Correspondence may also be addressed to Torsten Krude. Tel: 0044 1223 330111; Fax: 0044 1223 336676; Email: tk1{at}mole.bio.cam.ac.uk


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T. Krude, C. P. Christov, O. Hyrien, and K. Marheineke
Y RNA functions at the initiation step of mammalian chromosomal DNA replication
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C. P. Christov, T. J. Gardiner, D. Szuts, and T. Krude
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