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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

Variations of dimension values for the Spars1 MluI–EcoRI fragment as measured by the ‘Scanning adventure’ software with time (red trace). Successive frames are labelled from A to S. Because the first image captured is incomplete, the total length of A is not reported in graph A. Graph A is the variation of length before the formation of the right loop. Graph B is the variation of length after the formation of the loop (the software does not trace the DNA trajectory inside the loop).
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Figure 7: Variations of dimension values for the Spars1 MluI–EcoRI fragment as measured by the ‘Scanning adventure’ software with time (red trace). Successive frames are labelled from A to S. Because the first image captured is incomplete, the total length of A is not reported in graph A. Graph A is the variation of length before the formation of the right loop. Graph B is the variation of length after the formation of the loop (the software does not trace the DNA trajectory inside the loop).

Mentions: The AFM in situ analysis shows how even small changes in supercoiling can cause substantial structural modifications as, for instance, in the example shown in Supplementary Data S2 and analysed in Figures 6 and 7. In the series of recorded images, the permanent twisting and untwisting of the superhelix, necessary for it to explore different conformations while maintaining its two-dimensionality, make it possible to follow how the supercoiled organization generates a distant positive loop. The difference in length before looping (Figure 7A), and after looping (Figure 7B), indicates that the ‘blobby’ image on the molecule trajectory is really a loop. The distance between the start and the end of the loop indicate that the size was ∼130 bp. Topographical slices of the loop (insert, Figure 6) unambiguously show that it was right-handed. Four additional examples are shown (taken at the same location); in all cases the right-handedness of the newly formed loops is clear (bottom of Figure 6).Figure 7.


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)

Variations of dimension values for the Spars1 MluI–EcoRI fragment as measured by the ‘Scanning adventure’ software with time (red trace). Successive frames are labelled from A to S. Because the first image captured is incomplete, the total length of A is not reported in graph A. Graph A is the variation of length before the formation of the right loop. Graph B is the variation of length after the formation of the loop (the software does not trace the DNA trajectory inside the loop).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Variations of dimension values for the Spars1 MluI–EcoRI fragment as measured by the ‘Scanning adventure’ software with time (red trace). Successive frames are labelled from A to S. Because the first image captured is incomplete, the total length of A is not reported in graph A. Graph A is the variation of length before the formation of the right loop. Graph B is the variation of length after the formation of the loop (the software does not trace the DNA trajectory inside the loop).
Mentions: The AFM in situ analysis shows how even small changes in supercoiling can cause substantial structural modifications as, for instance, in the example shown in Supplementary Data S2 and analysed in Figures 6 and 7. In the series of recorded images, the permanent twisting and untwisting of the superhelix, necessary for it to explore different conformations while maintaining its two-dimensionality, make it possible to follow how the supercoiled organization generates a distant positive loop. The difference in length before looping (Figure 7A), and after looping (Figure 7B), indicates that the ‘blobby’ image on the molecule trajectory is really a loop. The distance between the start and the end of the loop indicate that the size was ∼130 bp. Topographical slices of the loop (insert, Figure 6) unambiguously show that it was right-handed. Four additional examples are shown (taken at the same location); in all cases the right-handedness of the newly formed loops is clear (bottom of Figure 6).Figure 7.

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