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Nucleic Acids Research, 2000, Vol. 28, No. 2 498-503
© 2000 Oxford University Press

Visualisation of plasmid replication intermediates containing reversed forks

E. Viguera, P. Hernández, D. B. Krimer, R. Lurz1 and J. B. Schvartzman*

Departamento de Biología Celular y del Desarrollo, CIB (CSIC), Velázquez 144, 28006 Madrid, Spain and 1Max-Planck-Institute for Molecular Genetics, Berlin, Germany

Blockage of replication forks can have deleterious consequences for the cell as it may prompt premature termination of DNA replication. Moreover, the blocked replication intermediate (RI) could be particularly sensitive to recombination processes. We analysed the different populations of RIs generated in vivo in the bacterial plasmid pPI21 after pausing of replication forks at the inversely oriented ColE1 origin. To achieve this goal, a new method was developed based on two-dimensional agarose gel electrophoresis. This method allows the isolation of specific RIs, even when they were rather scarce, from the total DNA. Here we describe the occurrence of RI restriction fragments containing reversed forks. These Holliday-like structures have been postulated but never observed before.

* To whom correspondence should be addressed. Tel: +34 91 564 4562; Fax: +34 91 564 8749; Email: schvartzman@cib.csic.es Present address: E. Viguera, Laboratoire de Génétique Microbienne, INRA, 78352 Jouy en Josas Cedex, France


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