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Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site.

Marilley M, Milani P, Thimonier J, Rocca-Serra J, Baldacci G - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT
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.

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Related in: MedlinePlus

Replication preinitiation model. (A) Poorly stable DNA with inherent structures made of curved and supercoiled features. (B) A positively supercoiled region (+) is recognized and bound by a component of pORC, Orc4p (red circle) which organizes the DNA into a negative loop (−). (C) A second site (+) is recognized and bound by a second Orc4p, and a second negative loop is generated (−). (D) These binding events cooperate in unwinding DNA and displace the apex of the curvature to the RI site. Although the region is not melted, strand opening at this site may be greatly facilitated (star).
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Figure 11: Replication preinitiation model. (A) Poorly stable DNA with inherent structures made of curved and supercoiled features. (B) A positively supercoiled region (+) is recognized and bound by a component of pORC, Orc4p (red circle) which organizes the DNA into a negative loop (−). (C) A second site (+) is recognized and bound by a second Orc4p, and a second negative loop is generated (−). (D) These binding events cooperate in unwinding DNA and displace the apex of the curvature to the RI site. Although the region is not melted, strand opening at this site may be greatly facilitated (star).

Mentions: We next looked on previous 2D gel electrophorectic studies of the ars2004 origin (3,31) and (23) and found that they did not refer to exactly the same segment of DNA. However, in both cases, it was shown that replication initiates within the analysed fragment. We found that the fragments studied overlap by ∼400 bp (Figure 11, red and blue lines), which may explain the similarity of their findings; it also suggested that the replication initiation site maps within these 400 bp. Modelling of the two candidate regions shows that the overlapping 400 bp region co-localizes exactly with the strongly curved DNA feature.Figure 11.


Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site.

Marilley M, Milani P, Thimonier J, Rocca-Serra J, Baldacci G - Nucleic Acids Res. (2007)

Replication preinitiation model. (A) Poorly stable DNA with inherent structures made of curved and supercoiled features. (B) A positively supercoiled region (+) is recognized and bound by a component of pORC, Orc4p (red circle) which organizes the DNA into a negative loop (−). (C) A second site (+) is recognized and bound by a second Orc4p, and a second negative loop is generated (−). (D) These binding events cooperate in unwinding DNA and displace the apex of the curvature to the RI site. Although the region is not melted, strand opening at this site may be greatly facilitated (star).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175326&req=5

Figure 11: Replication preinitiation model. (A) Poorly stable DNA with inherent structures made of curved and supercoiled features. (B) A positively supercoiled region (+) is recognized and bound by a component of pORC, Orc4p (red circle) which organizes the DNA into a negative loop (−). (C) A second site (+) is recognized and bound by a second Orc4p, and a second negative loop is generated (−). (D) These binding events cooperate in unwinding DNA and displace the apex of the curvature to the RI site. Although the region is not melted, strand opening at this site may be greatly facilitated (star).
Mentions: We next looked on previous 2D gel electrophorectic studies of the ars2004 origin (3,31) and (23) and found that they did not refer to exactly the same segment of DNA. However, in both cases, it was shown that replication initiates within the analysed fragment. We found that the fragments studied overlap by ∼400 bp (Figure 11, red and blue lines), which may explain the similarity of their findings; it also suggested that the replication initiation site maps within these 400 bp. Modelling of the two candidate regions shows that the overlapping 400 bp region co-localizes exactly with the strongly curved DNA feature.Figure 11.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus