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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

Analysis of the regions required for autonomous replication in ars 2004. (A and D) Modelling the region predicts right-handed superstructures co-mapping with regions I and III (3,25) essential for ars activity. Deletion of these regions also abolishes the structure (ΔI and ΔIII): see solid red line. Maximum of curvature is detected for phasing pitches of 8.9 and 6.1, respectively (window 200, step 2). (B) Modelling of region II shows a strong curvature; deletion of the region eliminates the structure (ΔII); see solid red line. Short deletions within the region II (ΔD, ΔE, ΔF), which were shown by Okuno to abolish or greatly reduce ars activity, alter the structure. (C) Replacing region II with a short A-stretch (A40) restores the activity and also clearly induces a strong curvature with identical positioning of the apex. (E) The 800-bp segment (blue line) that contains the RIP, identified by comparing 2D gel electrophoresis results (3), and the 835 bp fragment (red line), analysed in (23), that both contain a replication origin, are modelled. The two fragments overlap and the overlapping region corresponds precisely to the region of strong curvature that cannot be deleted without abolishing the ars function. (F) Mapping of the three regions (regions I, II and III) required for autonomous replication and enlargement (31). Thick black lines are essential regions. The open circle is the predicted opening site.
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Figure 9: Analysis of the regions required for autonomous replication in ars 2004. (A and D) Modelling the region predicts right-handed superstructures co-mapping with regions I and III (3,25) essential for ars activity. Deletion of these regions also abolishes the structure (ΔI and ΔIII): see solid red line. Maximum of curvature is detected for phasing pitches of 8.9 and 6.1, respectively (window 200, step 2). (B) Modelling of region II shows a strong curvature; deletion of the region eliminates the structure (ΔII); see solid red line. Short deletions within the region II (ΔD, ΔE, ΔF), which were shown by Okuno to abolish or greatly reduce ars activity, alter the structure. (C) Replacing region II with a short A-stretch (A40) restores the activity and also clearly induces a strong curvature with identical positioning of the apex. (E) The 800-bp segment (blue line) that contains the RIP, identified by comparing 2D gel electrophoresis results (3), and the 835 bp fragment (red line), analysed in (23), that both contain a replication origin, are modelled. The two fragments overlap and the overlapping region corresponds precisely to the region of strong curvature that cannot be deleted without abolishing the ars function. (F) Mapping of the three regions (regions I, II and III) required for autonomous replication and enlargement (31). Thick black lines are essential regions. The open circle is the predicted opening site.

Mentions: The RI in S. pombe ars2004 has been mapped to an 800-bp segment of DNA (3). Deletion and substitution studies have identified regions, called region I, region II and region III (Figure 9F), that are essential for replication function: the three regions appear to act in concert (31). We modelled the ars2004 element and found positive supercoils at positions corresponding to regions I and III, and a strong curvature at a position corresponding to region II. The model of an ars2004 from which these regions are deleted (ΔI, ΔII or ΔIII) shows the abolition of the structures (see red traces and compare with the reference curves in Figure 9A, B and D). This agrees well with Okuno's et al. results showing that these deletions abolish the ars activity (31).Figure 9.


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)

Analysis of the regions required for autonomous replication in ars 2004. (A and D) Modelling the region predicts right-handed superstructures co-mapping with regions I and III (3,25) essential for ars activity. Deletion of these regions also abolishes the structure (ΔI and ΔIII): see solid red line. Maximum of curvature is detected for phasing pitches of 8.9 and 6.1, respectively (window 200, step 2). (B) Modelling of region II shows a strong curvature; deletion of the region eliminates the structure (ΔII); see solid red line. Short deletions within the region II (ΔD, ΔE, ΔF), which were shown by Okuno to abolish or greatly reduce ars activity, alter the structure. (C) Replacing region II with a short A-stretch (A40) restores the activity and also clearly induces a strong curvature with identical positioning of the apex. (E) The 800-bp segment (blue line) that contains the RIP, identified by comparing 2D gel electrophoresis results (3), and the 835 bp fragment (red line), analysed in (23), that both contain a replication origin, are modelled. The two fragments overlap and the overlapping region corresponds precisely to the region of strong curvature that cannot be deleted without abolishing the ars function. (F) Mapping of the three regions (regions I, II and III) required for autonomous replication and enlargement (31). Thick black lines are essential regions. The open circle is the predicted opening site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 9: Analysis of the regions required for autonomous replication in ars 2004. (A and D) Modelling the region predicts right-handed superstructures co-mapping with regions I and III (3,25) essential for ars activity. Deletion of these regions also abolishes the structure (ΔI and ΔIII): see solid red line. Maximum of curvature is detected for phasing pitches of 8.9 and 6.1, respectively (window 200, step 2). (B) Modelling of region II shows a strong curvature; deletion of the region eliminates the structure (ΔII); see solid red line. Short deletions within the region II (ΔD, ΔE, ΔF), which were shown by Okuno to abolish or greatly reduce ars activity, alter the structure. (C) Replacing region II with a short A-stretch (A40) restores the activity and also clearly induces a strong curvature with identical positioning of the apex. (E) The 800-bp segment (blue line) that contains the RIP, identified by comparing 2D gel electrophoresis results (3), and the 835 bp fragment (red line), analysed in (23), that both contain a replication origin, are modelled. The two fragments overlap and the overlapping region corresponds precisely to the region of strong curvature that cannot be deleted without abolishing the ars function. (F) Mapping of the three regions (regions I, II and III) required for autonomous replication and enlargement (31). Thick black lines are essential regions. The open circle is the predicted opening site.
Mentions: The RI in S. pombe ars2004 has been mapped to an 800-bp segment of DNA (3). Deletion and substitution studies have identified regions, called region I, region II and region III (Figure 9F), that are essential for replication function: the three regions appear to act in concert (31). We modelled the ars2004 element and found positive supercoils at positions corresponding to regions I and III, and a strong curvature at a position corresponding to region II. The model of an ars2004 from which these regions are deleted (ΔI, ΔII or ΔIII) shows the abolition of the structures (see red traces and compare with the reference curves in Figure 9A, B and D). This agrees well with Okuno's et al. results showing that these deletions abolish the ars activity (31).Figure 9.

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