Nucleic Acids Research Advance Access originally published online on October 11, 2007
Nucleic Acids Research 2007 35(20):6832-6845; doi:10.1093/nar/gkm733
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Nucleic Acids Research, 2007, Vol. 35, No. 20 6832-6845
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site
1Régulation génique et fonctionnelle & microscopie champ proche, EA 3290, IFR 125, Faculté de Médecine, Université de la Méditerranée, 27 Bd Jean Moulin, 13385 Marseille cedex 5 and 2Genotoxicologie et cycle cellulaire, UMR 2027, Institut Curie-CNRS, Centre universitaire, Bât. 110, 91405 Orsay, France
* To whom correspondence should be addressed. Tel/Fax: +33 (0)4 91 79 48 60; Email: monique.marilley{at}medecine.univ-mrs.fr
Received April 16, 2007. Revised September 3, 2007. Accepted September 4, 2007.
The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.