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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

(A) Curvature map along Spars1 MluI–EcoRI fragment. A strong curvature is predicted. Bolshoy dinucleotide parameters (17) were used for DNA modelling. The position of the RI site is indicated. The window is 150 bp, and each step 1 bp. (B) AFM imaging in liquid and Spars1 trajectory analysis. ENDS ratio variation profile. Window size is 150 bp. Mean value and SD are indicated. A strong curvature is clearly visible. Position of the RI site is indicated.
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Figure 4: (A) Curvature map along Spars1 MluI–EcoRI fragment. A strong curvature is predicted. Bolshoy dinucleotide parameters (17) were used for DNA modelling. The position of the RI site is indicated. The window is 150 bp, and each step 1 bp. (B) AFM imaging in liquid and Spars1 trajectory analysis. ENDS ratio variation profile. Window size is 150 bp. Mean value and SD are indicated. A strong curvature is clearly visible. Position of the RI site is indicated.

Mentions: Using Bolshoy values (17), the DNA path of the Spars1 fragment was calculated as the result of one translation and three rotations (roll, tilt and twist) at each base step (Figure 4A). The RI site (365–368 bp) was mapped in the intrinsically curved region nearly 80 bp from the apex.Figure 4.


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)

(A) Curvature map along Spars1 MluI–EcoRI fragment. A strong curvature is predicted. Bolshoy dinucleotide parameters (17) were used for DNA modelling. The position of the RI site is indicated. The window is 150 bp, and each step 1 bp. (B) AFM imaging in liquid and Spars1 trajectory analysis. ENDS ratio variation profile. Window size is 150 bp. Mean value and SD are indicated. A strong curvature is clearly visible. Position of the RI site is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: (A) Curvature map along Spars1 MluI–EcoRI fragment. A strong curvature is predicted. Bolshoy dinucleotide parameters (17) were used for DNA modelling. The position of the RI site is indicated. The window is 150 bp, and each step 1 bp. (B) AFM imaging in liquid and Spars1 trajectory analysis. ENDS ratio variation profile. Window size is 150 bp. Mean value and SD are indicated. A strong curvature is clearly visible. Position of the RI site is indicated.
Mentions: Using Bolshoy values (17), the DNA path of the Spars1 fragment was calculated as the result of one translation and three rotations (roll, tilt and twist) at each base step (Figure 4A). The RI site (365–368 bp) was mapped in the intrinsically curved region nearly 80 bp from the apex.Figure 4.

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