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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

Formation of a right-handed loop. Images 1, 2 and 3 are AFM images of the MluI-EcoRI segment taken 0, 5 and 7 min, respectively, after the beginning of the observation. The red rectangles correspond to the enlarged regions beneath. White arrows correspond to the superhelix pitch. This pitch increases or decreases with time. Image number 3 shows the newly formed supercoiled loop. Insert: height variation on successive slices (right) corresponding to measurements on the loop as described in the scheme (left). These unambiguously show the positive organization of this loop. The loop size is ∼130 bp. Last line, α, β, γ, δ are four independent examples of right-handed loops at the same position on the molecule. One of them, loop (β), formed before the first scan, is larger.
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Figure 6: Formation of a right-handed loop. Images 1, 2 and 3 are AFM images of the MluI-EcoRI segment taken 0, 5 and 7 min, respectively, after the beginning of the observation. The red rectangles correspond to the enlarged regions beneath. White arrows correspond to the superhelix pitch. This pitch increases or decreases with time. Image number 3 shows the newly formed supercoiled loop. Insert: height variation on successive slices (right) corresponding to measurements on the loop as described in the scheme (left). These unambiguously show the positive organization of this loop. The loop size is ∼130 bp. Last line, α, β, γ, δ are four independent examples of right-handed loops at the same position on the molecule. One of them, loop (β), formed before the first scan, is larger.

Mentions: We used AFM imaging in liquid to validate our theoretical predictions: this technique revealed both the strong curvature and the supercoiled right-handed organization (Figure 6). The positions of this latter structure was measured in nanometre and converted into base pair: the superhelical structure was found to encompass ∼200 bp (approximately from position 80 to 290 bp of the MluI end of the DNA fragment).Figure 6.


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)

Formation of a right-handed loop. Images 1, 2 and 3 are AFM images of the MluI-EcoRI segment taken 0, 5 and 7 min, respectively, after the beginning of the observation. The red rectangles correspond to the enlarged regions beneath. White arrows correspond to the superhelix pitch. This pitch increases or decreases with time. Image number 3 shows the newly formed supercoiled loop. Insert: height variation on successive slices (right) corresponding to measurements on the loop as described in the scheme (left). These unambiguously show the positive organization of this loop. The loop size is ∼130 bp. Last line, α, β, γ, δ are four independent examples of right-handed loops at the same position on the molecule. One of them, loop (β), formed before the first scan, is larger.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Formation of a right-handed loop. Images 1, 2 and 3 are AFM images of the MluI-EcoRI segment taken 0, 5 and 7 min, respectively, after the beginning of the observation. The red rectangles correspond to the enlarged regions beneath. White arrows correspond to the superhelix pitch. This pitch increases or decreases with time. Image number 3 shows the newly formed supercoiled loop. Insert: height variation on successive slices (right) corresponding to measurements on the loop as described in the scheme (left). These unambiguously show the positive organization of this loop. The loop size is ∼130 bp. Last line, α, β, γ, δ are four independent examples of right-handed loops at the same position on the molecule. One of them, loop (β), formed before the first scan, is larger.
Mentions: We used AFM imaging in liquid to validate our theoretical predictions: this technique revealed both the strong curvature and the supercoiled right-handed organization (Figure 6). The positions of this latter structure was measured in nanometre and converted into base pair: the superhelical structure was found to encompass ∼200 bp (approximately from position 80 to 290 bp of the MluI end of the DNA fragment).Figure 6.

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