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Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication.

Ruiz-Masó JA, Lurz R, Espinosa M, del Solar G - Nucleic Acids Res. (2007)

Bottom Line: Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus.On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus.This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC. Ramiro de Maeztu, 9. E-28040-Madrid, Spain.

ABSTRACT
Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

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High resolution contacts of RepB with the entire dso (A) and with the nic locus (B). DNAs were 5′-end labelled and incubated with RepB. The complexes generated were treated with HO• (A and B) or with DMS (B) as described in ‘Materials and Methods’. Sequencing gels show the modification patterns of the 5′ regions of both strands. Absorption scans of naked DNAs (black lines) and complexes C1 (red lines) and C2 (blue lines) are displayed between the nucleotide sequence and the corresponding gel. To facilitate the detection of weak protections in B, the scans corresponding to the naked and complexed DNAs are shown superimposed around the footprints. PDR and DDR are shadowed. The IR-I element is displayed with its left (L) and right (R) arms. The dash shows the position of the nick site. Bases whose deoxyriboses are protected by RepB from HO• cleavage are indicated by triangles of different sizes (related to the extent of protection). In B, bases hyperexposed (black circles) or protected (open circles) by RepB against methylation with DMS are also shown.
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Figure 2: High resolution contacts of RepB with the entire dso (A) and with the nic locus (B). DNAs were 5′-end labelled and incubated with RepB. The complexes generated were treated with HO• (A and B) or with DMS (B) as described in ‘Materials and Methods’. Sequencing gels show the modification patterns of the 5′ regions of both strands. Absorption scans of naked DNAs (black lines) and complexes C1 (red lines) and C2 (blue lines) are displayed between the nucleotide sequence and the corresponding gel. To facilitate the detection of weak protections in B, the scans corresponding to the naked and complexed DNAs are shown superimposed around the footprints. PDR and DDR are shadowed. The IR-I element is displayed with its left (L) and right (R) arms. The dash shows the position of the nick site. Bases whose deoxyriboses are protected by RepB from HO• cleavage are indicated by triangles of different sizes (related to the extent of protection). In B, bases hyperexposed (black circles) or protected (open circles) by RepB against methylation with DMS are also shown.

Mentions: Analysis of the contacts between RepB and its target DNAs were performed by HO• and DMS footprinting of complexes C1 and C2 generated by binding of the protein to the DNA fragments containing the nic locus, the bind locus, or the entire dso. The DNAs were 5′-end labelled (top or bottom strand) and modified either prior to (HO• assays) or after (DMS assays) separation of free and RepB-complexed DNAs. The results are summarized in Figure 2A and B.Figure 2.


Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication.

Ruiz-Masó JA, Lurz R, Espinosa M, del Solar G - Nucleic Acids Res. (2007)

High resolution contacts of RepB with the entire dso (A) and with the nic locus (B). DNAs were 5′-end labelled and incubated with RepB. The complexes generated were treated with HO• (A and B) or with DMS (B) as described in ‘Materials and Methods’. Sequencing gels show the modification patterns of the 5′ regions of both strands. Absorption scans of naked DNAs (black lines) and complexes C1 (red lines) and C2 (blue lines) are displayed between the nucleotide sequence and the corresponding gel. To facilitate the detection of weak protections in B, the scans corresponding to the naked and complexed DNAs are shown superimposed around the footprints. PDR and DDR are shadowed. The IR-I element is displayed with its left (L) and right (R) arms. The dash shows the position of the nick site. Bases whose deoxyriboses are protected by RepB from HO• cleavage are indicated by triangles of different sizes (related to the extent of protection). In B, bases hyperexposed (black circles) or protected (open circles) by RepB against methylation with DMS are also shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851628&req=5

Figure 2: High resolution contacts of RepB with the entire dso (A) and with the nic locus (B). DNAs were 5′-end labelled and incubated with RepB. The complexes generated were treated with HO• (A and B) or with DMS (B) as described in ‘Materials and Methods’. Sequencing gels show the modification patterns of the 5′ regions of both strands. Absorption scans of naked DNAs (black lines) and complexes C1 (red lines) and C2 (blue lines) are displayed between the nucleotide sequence and the corresponding gel. To facilitate the detection of weak protections in B, the scans corresponding to the naked and complexed DNAs are shown superimposed around the footprints. PDR and DDR are shadowed. The IR-I element is displayed with its left (L) and right (R) arms. The dash shows the position of the nick site. Bases whose deoxyriboses are protected by RepB from HO• cleavage are indicated by triangles of different sizes (related to the extent of protection). In B, bases hyperexposed (black circles) or protected (open circles) by RepB against methylation with DMS are also shown.
Mentions: Analysis of the contacts between RepB and its target DNAs were performed by HO• and DMS footprinting of complexes C1 and C2 generated by binding of the protein to the DNA fragments containing the nic locus, the bind locus, or the entire dso. The DNAs were 5′-end labelled (top or bottom strand) and modified either prior to (HO• assays) or after (DMS assays) separation of free and RepB-complexed DNAs. The results are summarized in Figure 2A and B.Figure 2.

Bottom Line: Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus.On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus.This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC. Ramiro de Maeztu, 9. E-28040-Madrid, Spain.

ABSTRACT
Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

Show MeSH
Related in: MedlinePlus