Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site.
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.
Marilley
Monique
M
Régulation 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, France. monique.marilley@medecine.univ-mrs.fr
Milani
Pascale
P
Thimonier
Jean
J
Rocca-Serra
José
J
Baldacci
Giuseppe
G
D000818:Animals; D004261:DNA Replication; D004330:Drosophila / Q000235:genetics; D004527:Egg Proteins / Q000235:genetics; D007700:Kinetics; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D051738:Origin Recognition Complex; D018741:Replication Origin; D012568:Schizosaccharomyces / Q000235:genetics* / Q000648:ultrastructure*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics
D004527:Egg Proteins; D051738:Origin Recognition Complex; D029702:Schizosaccharomyces pombe Proteins; C015753:chorion proteins