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J. Biol. Chem., Vol. 282, Issue 25, 18190-18196, June 22, 2007

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Replication Fork Reversal Occurs Spontaneously after Digestion but Is Constrained in Supercoiled Domains^*

From the Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain

Replication fork reversal was investigated in undigested and linearized replication intermediates of bacterial DNA plasmids containing a stalled fork. Two-dimensional agarose gel electrophoresis, a branch migration and extrusion assay, electron microscopy, and DNA-psoralen cross-linking were used to show that extensive replication fork reversal and extrusion of the nascent-nascent duplex occurs spontaneously after DNA nicking and restriction enzyme digestion but that fork retreat is severely limited in covalently closed supercoiled domains.

Received for publication, February 21, 2007 , and in revised form, April 23, 2007.

^* This work was supported by Grants BIO2005-02224 (to J. B. S.) and BFU2004-00125 to PH from the Spanish Ministerio de Educación y Ciencia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 34-91-837-3112; Fax: 34-91-536-0432; E-mail: schvartzman{at}cib.csic.es.

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