Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Restriction sites in pART1. (B) The EcoRI–EcoRI 1204 bp insert was digested with MluI and labelled. Positions of segments ars1-4 and ars1-3 according to Ref. (11) are indicated. The region reported as essential for ARS activity by Ref. (28) is also indicated. RI is the site of initiation of replication described in Ref. (4). The pORC binding position according to Ref. (9) is indicated. The dotted arrow shows the direction of transcription. (C) AFM imaging in liquid of a labelled molecule and localization of the RI site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175326&req=5

Figure 1: (A) Restriction sites in pART1. (B) The EcoRI–EcoRI 1204 bp insert was digested with MluI and labelled. Positions of segments ars1-4 and ars1-3 according to Ref. (11) are indicated. The region reported as essential for ARS activity by Ref. (28) is also indicated. RI is the site of initiation of replication described in Ref. (4). The pORC binding position according to Ref. (9) is indicated. The dotted arrow shows the direction of transcription. (C) AFM imaging in liquid of a labelled molecule and localization of the RI site.

Mentions: The Spars1 fragment (1204 bp) was obtained by EcoRI digestion of pART1 (7278 bp) and further digested with MluI for DNA orientation by labelling of the EcoRI end (Figure 1). ORI regions in S. pombe map in A+T-rich islands (23). Thus, A+T-richness may facilitate strand separation at the origin, but it may also generate some particular base composition required for the formation of various structural features. We, therefore, examined first the thermodynamic stability of the region, and then analysed the different structures featuring Spars1 DNA. Our studies include both a predictive and an experimental approach.Figure 1.


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) Restriction sites in pART1. (B) The EcoRI–EcoRI 1204 bp insert was digested with MluI and labelled. Positions of segments ars1-4 and ars1-3 according to Ref. (11) are indicated. The region reported as essential for ARS activity by Ref. (28) is also indicated. RI is the site of initiation of replication described in Ref. (4). The pORC binding position according to Ref. (9) is indicated. The dotted arrow shows the direction of transcription. (C) AFM imaging in liquid of a labelled molecule and localization of the RI site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: (A) Restriction sites in pART1. (B) The EcoRI–EcoRI 1204 bp insert was digested with MluI and labelled. Positions of segments ars1-4 and ars1-3 according to Ref. (11) are indicated. The region reported as essential for ARS activity by Ref. (28) is also indicated. RI is the site of initiation of replication described in Ref. (4). The pORC binding position according to Ref. (9) is indicated. The dotted arrow shows the direction of transcription. (C) AFM imaging in liquid of a labelled molecule and localization of the RI site.
Mentions: The Spars1 fragment (1204 bp) was obtained by EcoRI digestion of pART1 (7278 bp) and further digested with MluI for DNA orientation by labelling of the EcoRI end (Figure 1). ORI regions in S. pombe map in A+T-rich islands (23). Thus, A+T-richness may facilitate strand separation at the origin, but it may also generate some particular base composition required for the formation of various structural features. We, therefore, examined first the thermodynamic stability of the region, and then analysed the different structures featuring Spars1 DNA. Our studies include both a predictive and an experimental approach.Figure 1.

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