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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 bind locus. (A) DMS footprint of RepB bound to a linear DNA fragment containing the DDR. Bases hypermethylated (grey or black triangles, depending on whether there is a slight or a marked increase, respectively, in the DMS sensitivity) or protected (red triangles) in each strand by bound RepB are shown. To the right, the DNA sequence of the footprinted regions is displayed with its 5′→3′ directionality. Lanes: F, free DNA; C1, DNA of complex C1; G+A, Maxam and Gilbert sequencing ladder for purines. (B) Scheme of the B-DNA double helix of the DDR displayed with the nucleotide sequences of the top and bottom strands. Bases whose deoxyriboses are protected by RepB against HO• attack are shown in boldface letters or as green-shadowed regions on the double helix. Bases which become hypermethylated (grey/black triangles, black-lined encircled letters) or protected from methylation (red triangles, red-lined encircled letters) upon binding of RepB are indicated on the sequence and on the DNA double helix. The three repeats constituting the DDR are indicated by arrows. (C) DMS footprint of RepB bound to the DDR on supercoiled pMV158. The methylation sites on the top strand were mapped by primer extension using labelled primer dso4 corresponding to the bottom strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). The methylation pattern of the bind locus was obtained in the absence of RepB or at the indicated concentrations of the protein. To the right, the DNA sequence at the footprinted regions is displayed with its 5′→3′ directionality. The same symbol code as in A was used.
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Figure 3: High resolution contacts of RepB with the bind locus. (A) DMS footprint of RepB bound to a linear DNA fragment containing the DDR. Bases hypermethylated (grey or black triangles, depending on whether there is a slight or a marked increase, respectively, in the DMS sensitivity) or protected (red triangles) in each strand by bound RepB are shown. To the right, the DNA sequence of the footprinted regions is displayed with its 5′→3′ directionality. Lanes: F, free DNA; C1, DNA of complex C1; G+A, Maxam and Gilbert sequencing ladder for purines. (B) Scheme of the B-DNA double helix of the DDR displayed with the nucleotide sequences of the top and bottom strands. Bases whose deoxyriboses are protected by RepB against HO• attack are shown in boldface letters or as green-shadowed regions on the double helix. Bases which become hypermethylated (grey/black triangles, black-lined encircled letters) or protected from methylation (red triangles, red-lined encircled letters) upon binding of RepB are indicated on the sequence and on the DNA double helix. The three repeats constituting the DDR are indicated by arrows. (C) DMS footprint of RepB bound to the DDR on supercoiled pMV158. The methylation sites on the top strand were mapped by primer extension using labelled primer dso4 corresponding to the bottom strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). The methylation pattern of the bind locus was obtained in the absence of RepB or at the indicated concentrations of the protein. To the right, the DNA sequence at the footprinted regions is displayed with its 5′→3′ directionality. The same symbol code as in A was used.

Mentions: The footprints mediated by RepB in the separate bind locus refine our previous finding that the DDR constitute the primary binding site of the initiator (18). Changes in the DMS reactivity profiles upon RepB binding were analysed for each strand of the DDR (Figure 3A and B). The results showed that RepB interacts through the major groove of the DNA with the same three Gs of each direct repeat, protecting them against methylation. Enhanced DMS reactivity of some Gs and Cs in each repeat constituting the DDR was also observed. Since DMS reactivity of C residues results from local DNA conformational changes such as unwinding or distortion of the double helix (32), we tested whether binding of RepB induced strand openings. To do this, the same RepB-bind C1 complex was subjected to KMnO4 probing, but no local melting of the DNA strands could be observed (not shown). The HO• and DMS reactivity profiles of the pMV158 bind locus are displayed on the DNA double helix in Figure 3B. They reflect that the interactions of RepB with the DDR involve only one face of the double helix, generating a significant distortion on it. Furthermore, a clear repeat profile was observed for the protein bound to the bind locus (Figure 3), which suggests that RepB (most likely in its hexameric form) employs three identical DNA-binding motifs to bind to the DDR. When, instead of linear DNA, supercoiled pMV158 DNA was used as the binding substrate, essentially the same DMS reactivity profile of the DDR region was observed (the methylation pattern of the top strand as deduced from stops in the extension of the bottom strand is shown in Figure 3C). This was expected as the DNA of the bind locus is predicted to have similar conformation in linear and supercoiled molecules.Figure 3.


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 bind locus. (A) DMS footprint of RepB bound to a linear DNA fragment containing the DDR. Bases hypermethylated (grey or black triangles, depending on whether there is a slight or a marked increase, respectively, in the DMS sensitivity) or protected (red triangles) in each strand by bound RepB are shown. To the right, the DNA sequence of the footprinted regions is displayed with its 5′→3′ directionality. Lanes: F, free DNA; C1, DNA of complex C1; G+A, Maxam and Gilbert sequencing ladder for purines. (B) Scheme of the B-DNA double helix of the DDR displayed with the nucleotide sequences of the top and bottom strands. Bases whose deoxyriboses are protected by RepB against HO• attack are shown in boldface letters or as green-shadowed regions on the double helix. Bases which become hypermethylated (grey/black triangles, black-lined encircled letters) or protected from methylation (red triangles, red-lined encircled letters) upon binding of RepB are indicated on the sequence and on the DNA double helix. The three repeats constituting the DDR are indicated by arrows. (C) DMS footprint of RepB bound to the DDR on supercoiled pMV158. The methylation sites on the top strand were mapped by primer extension using labelled primer dso4 corresponding to the bottom strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). The methylation pattern of the bind locus was obtained in the absence of RepB or at the indicated concentrations of the protein. To the right, the DNA sequence at the footprinted regions is displayed with its 5′→3′ directionality. The same symbol code as in A was used.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 3: High resolution contacts of RepB with the bind locus. (A) DMS footprint of RepB bound to a linear DNA fragment containing the DDR. Bases hypermethylated (grey or black triangles, depending on whether there is a slight or a marked increase, respectively, in the DMS sensitivity) or protected (red triangles) in each strand by bound RepB are shown. To the right, the DNA sequence of the footprinted regions is displayed with its 5′→3′ directionality. Lanes: F, free DNA; C1, DNA of complex C1; G+A, Maxam and Gilbert sequencing ladder for purines. (B) Scheme of the B-DNA double helix of the DDR displayed with the nucleotide sequences of the top and bottom strands. Bases whose deoxyriboses are protected by RepB against HO• attack are shown in boldface letters or as green-shadowed regions on the double helix. Bases which become hypermethylated (grey/black triangles, black-lined encircled letters) or protected from methylation (red triangles, red-lined encircled letters) upon binding of RepB are indicated on the sequence and on the DNA double helix. The three repeats constituting the DDR are indicated by arrows. (C) DMS footprint of RepB bound to the DDR on supercoiled pMV158. The methylation sites on the top strand were mapped by primer extension using labelled primer dso4 corresponding to the bottom strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). The methylation pattern of the bind locus was obtained in the absence of RepB or at the indicated concentrations of the protein. To the right, the DNA sequence at the footprinted regions is displayed with its 5′→3′ directionality. The same symbol code as in A was used.
Mentions: The footprints mediated by RepB in the separate bind locus refine our previous finding that the DDR constitute the primary binding site of the initiator (18). Changes in the DMS reactivity profiles upon RepB binding were analysed for each strand of the DDR (Figure 3A and B). The results showed that RepB interacts through the major groove of the DNA with the same three Gs of each direct repeat, protecting them against methylation. Enhanced DMS reactivity of some Gs and Cs in each repeat constituting the DDR was also observed. Since DMS reactivity of C residues results from local DNA conformational changes such as unwinding or distortion of the double helix (32), we tested whether binding of RepB induced strand openings. To do this, the same RepB-bind C1 complex was subjected to KMnO4 probing, but no local melting of the DNA strands could be observed (not shown). The HO• and DMS reactivity profiles of the pMV158 bind locus are displayed on the DNA double helix in Figure 3B. They reflect that the interactions of RepB with the DDR involve only one face of the double helix, generating a significant distortion on it. Furthermore, a clear repeat profile was observed for the protein bound to the bind locus (Figure 3), which suggests that RepB (most likely in its hexameric form) employs three identical DNA-binding motifs to bind to the DDR. When, instead of linear DNA, supercoiled pMV158 DNA was used as the binding substrate, essentially the same DMS reactivity profile of the DDR region was observed (the methylation pattern of the top strand as deduced from stops in the extension of the bottom strand is shown in Figure 3C). This was expected as the DNA of the bind locus is predicted to have similar conformation in linear and supercoiled molecules.Figure 3.

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