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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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Presence of curved and supercoiled organization in a Drosophila replication origin: analysis of the DNA region containing Ace 3 and ori-beta in the Drosophila genome. (A) Unusual structures co-map with ACE3 and ori-beta. (B) Curvature map of reference: a strong curved region is associated with ACE3. (C) An evolutionarily conserved 142 bp core sequence (red trace) maps with right-handed DNA. (D) The beta-region described in Ref. (35), as a required component for amplification function (red trace) maps with a superhelical DNA (negative superhelicity). The direction of S-18 chorion gene transcription is indicated. Phasing is between 8 and 14 with steps of 0.25. The window is 150 bp, and each step is 2 bp.
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Figure 10: Presence of curved and supercoiled organization in a Drosophila replication origin: analysis of the DNA region containing Ace 3 and ori-beta in the Drosophila genome. (A) Unusual structures co-map with ACE3 and ori-beta. (B) Curvature map of reference: a strong curved region is associated with ACE3. (C) An evolutionarily conserved 142 bp core sequence (red trace) maps with right-handed DNA. (D) The beta-region described in Ref. (35), as a required component for amplification function (red trace) maps with a superhelical DNA (negative superhelicity). The direction of S-18 chorion gene transcription is indicated. Phasing is between 8 and 14 with steps of 0.25. The window is 150 bp, and each step is 2 bp.

Mentions: In the chorion gene locus of Drosophila, the amplification control element 3 (ACE3) functions as a replicator and the 3800 bp SalI fragment containing it has been studied in great detail. This segment contains two striking sequence elements, the alpha region upstream from the S18 chorion gene and the beta region downstream from it. Recently, the 320 bp ACE3 sequence element (34) was shown to contain an evolutionarily conserved 142 bp core region, and deletion mapping of the 840 bp ori-beta identified a required region of 226 bp that was called the beta region (35). We modelled the 3800-bp segment and found two regions of unusual structural features including curved and inherently supercoiled DNAs and also found that they coincide with the 320 and 840 bp segments (Figure 10A and B). Moreover, the essential and evolutionarily conserved core region corresponds exactly to the predicted structure (Figure 10C) whereas the ‘beta-region’ co-maps with the supercoiled DNA (Figure 10D). Note that an analysis of the localization of the DmORC protein complex showed the influence of DNA torsion stress on its binding (10): in vivo, the 320 bp ACE3 fragment is sufficient to localize DmORC in follicle cells. This is also the case for ori-beta (36).Figure 10.


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)

Presence of curved and supercoiled organization in a Drosophila replication origin: analysis of the DNA region containing Ace 3 and ori-beta in the Drosophila genome. (A) Unusual structures co-map with ACE3 and ori-beta. (B) Curvature map of reference: a strong curved region is associated with ACE3. (C) An evolutionarily conserved 142 bp core sequence (red trace) maps with right-handed DNA. (D) The beta-region described in Ref. (35), as a required component for amplification function (red trace) maps with a superhelical DNA (negative superhelicity). The direction of S-18 chorion gene transcription is indicated. Phasing is between 8 and 14 with steps of 0.25. The window is 150 bp, and each step is 2 bp.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 10: Presence of curved and supercoiled organization in a Drosophila replication origin: analysis of the DNA region containing Ace 3 and ori-beta in the Drosophila genome. (A) Unusual structures co-map with ACE3 and ori-beta. (B) Curvature map of reference: a strong curved region is associated with ACE3. (C) An evolutionarily conserved 142 bp core sequence (red trace) maps with right-handed DNA. (D) The beta-region described in Ref. (35), as a required component for amplification function (red trace) maps with a superhelical DNA (negative superhelicity). The direction of S-18 chorion gene transcription is indicated. Phasing is between 8 and 14 with steps of 0.25. The window is 150 bp, and each step is 2 bp.
Mentions: In the chorion gene locus of Drosophila, the amplification control element 3 (ACE3) functions as a replicator and the 3800 bp SalI fragment containing it has been studied in great detail. This segment contains two striking sequence elements, the alpha region upstream from the S18 chorion gene and the beta region downstream from it. Recently, the 320 bp ACE3 sequence element (34) was shown to contain an evolutionarily conserved 142 bp core region, and deletion mapping of the 840 bp ori-beta identified a required region of 226 bp that was called the beta region (35). We modelled the 3800-bp segment and found two regions of unusual structural features including curved and inherently supercoiled DNAs and also found that they coincide with the 320 and 840 bp segments (Figure 10A and B). Moreover, the essential and evolutionarily conserved core region corresponds exactly to the predicted structure (Figure 10C) whereas the ‘beta-region’ co-maps with the supercoiled DNA (Figure 10D). Note that an analysis of the localization of the DmORC protein complex showed the influence of DNA torsion stress on its binding (10): in vivo, the 320 bp ACE3 fragment is sufficient to localize DmORC in follicle cells. This is also the case for ori-beta (36).Figure 10.

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