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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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DMS and KMnO4 footprints of RepB bound to the nic locus on supercoiled DNAs. (A) Sequencing gels displaying DMS sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, pCGA7, a pC194 derivative which bears the nic region of the pMV158 dso was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described under ‘Materials and Methods’. RepB concentrations were 0.36, 0.6, 1.2 and 1.8 µM (lanes 1–4) for the reactions containing pMV158 DNA, and 0.6, 1.2, 1.8 and 4.8 M (lanes 5–8) for the pCGA7 DNA. The methylation sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of IR-I, with its left (L) and right (R) arms and its central region harbouring the nick sequence, is shown. To the right, the DNA sequence of the bottom strand at the footprints is displayed with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are indicated with red boxes. The same bands in pCGA7 are in orange boxes. (B) Quantification of the experiment shown in A by PhosphorImager analysis using Quantity One software. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 of pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (red line for pMV158 and orange line for pCGA7) are superimposed. Bases whose DMS sensitivity is modified in the presence of RepB are indicated with red (pMV158) or orange (pCGA7) bars. Bar heights are proportional to the percentage of DMS sensitivity enhancement. (C) Sequencing gels displaying KMnO4 sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, plasmid pCGA7 was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described in ‘Materials and Methods’. RepB concentrations were 0.6 µM (lanes 1 and 5), 1.8 µM (lanes 2 and 6), 2.4 µM (lanes 3 and 7) and 4.8 µM (lanes 4 and 8). The modification sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of the central region and right arm (R) of IR-I is displayed. To the right, the DNA sequence of the bottom strand at the distorted DNA regions is shown with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are within blue boxes. The same bands in pCGA7 are within green boxes. The arrows point to sites of high RepB-independent KMnO4 reactivity. (D) Quantification of the experiment shown in C. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 corresponding to pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (blue line for pMV158 and green line for pCGA7) are superimposed. Bases whose KMnO4 sensitivity is modified in the presence of RepB are indicated with blue (pMV158) or green (pCGA7) bars. Bar heights are proportional to the percentage of enhancement in the KMnO4 reactivity. (E) Sequencing gels displaying KMnO4 sensitivity of the bottom strand close to the bind region, in the absence and in the presence of RepB. Supercoiled pMV158 DNA was incubated without RepB (–) or in the presence of 0.6, 1.8, 2.4 and 4.8 µM of the protein (lanes 1–4, respectively). KMnO4 reaction and mapping of the modification sites was as in C. A dideoxy sequencing ladder (lanes A, C, G, T) was included as a control. To the right, the DNA sequence of the bottom strand at the DNA region distorted upon binding of RepB to the nic locus is displayed with its 5′→3′ directionality. Bands whose intensity is modified in the presence of RepB are within a blue box. (F) Quantification of the experiment shown in ahown in E. Scans of lanes (–) (black line) and 4 (blue line) are drawn superimposed. Bases that increase their KMnO4 sensitivity in the presence of RepB are indicated with blue bars whose heights are proportional to the percentage of enhancement of the KMnO4 reactivity. (G) Scheme of the bottom strand sites of the pMV158 dso whose DMS or KMnO4 sensitivity is modified upon binding of RepB to the nic locus. Once RepB is bound to its high affinity target, the bind locus, the protein would bind to its lower affinity target in the nic locus, then rendering some sites highly sensitive to DMS (red ellipses) or to KMnO4 (blue ellipses). Whereas most of the sites that increase their KMnO4 sensitivity are within the nic locus, the most distorted site was found close to the DDR. The IR-I cruciform of the pMV158 dso is represented since binding of RepB to the nic locus promotes its extrusion.
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Figure 7: DMS and KMnO4 footprints of RepB bound to the nic locus on supercoiled DNAs. (A) Sequencing gels displaying DMS sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, pCGA7, a pC194 derivative which bears the nic region of the pMV158 dso was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described under ‘Materials and Methods’. RepB concentrations were 0.36, 0.6, 1.2 and 1.8 µM (lanes 1–4) for the reactions containing pMV158 DNA, and 0.6, 1.2, 1.8 and 4.8 M (lanes 5–8) for the pCGA7 DNA. The methylation sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of IR-I, with its left (L) and right (R) arms and its central region harbouring the nick sequence, is shown. To the right, the DNA sequence of the bottom strand at the footprints is displayed with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are indicated with red boxes. The same bands in pCGA7 are in orange boxes. (B) Quantification of the experiment shown in A by PhosphorImager analysis using Quantity One software. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 of pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (red line for pMV158 and orange line for pCGA7) are superimposed. Bases whose DMS sensitivity is modified in the presence of RepB are indicated with red (pMV158) or orange (pCGA7) bars. Bar heights are proportional to the percentage of DMS sensitivity enhancement. (C) Sequencing gels displaying KMnO4 sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, plasmid pCGA7 was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described in ‘Materials and Methods’. RepB concentrations were 0.6 µM (lanes 1 and 5), 1.8 µM (lanes 2 and 6), 2.4 µM (lanes 3 and 7) and 4.8 µM (lanes 4 and 8). The modification sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of the central region and right arm (R) of IR-I is displayed. To the right, the DNA sequence of the bottom strand at the distorted DNA regions is shown with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are within blue boxes. The same bands in pCGA7 are within green boxes. The arrows point to sites of high RepB-independent KMnO4 reactivity. (D) Quantification of the experiment shown in C. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 corresponding to pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (blue line for pMV158 and green line for pCGA7) are superimposed. Bases whose KMnO4 sensitivity is modified in the presence of RepB are indicated with blue (pMV158) or green (pCGA7) bars. Bar heights are proportional to the percentage of enhancement in the KMnO4 reactivity. (E) Sequencing gels displaying KMnO4 sensitivity of the bottom strand close to the bind region, in the absence and in the presence of RepB. Supercoiled pMV158 DNA was incubated without RepB (–) or in the presence of 0.6, 1.8, 2.4 and 4.8 µM of the protein (lanes 1–4, respectively). KMnO4 reaction and mapping of the modification sites was as in C. A dideoxy sequencing ladder (lanes A, C, G, T) was included as a control. To the right, the DNA sequence of the bottom strand at the DNA region distorted upon binding of RepB to the nic locus is displayed with its 5′→3′ directionality. Bands whose intensity is modified in the presence of RepB are within a blue box. (F) Quantification of the experiment shown in ahown in E. Scans of lanes (–) (black line) and 4 (blue line) are drawn superimposed. Bases that increase their KMnO4 sensitivity in the presence of RepB are indicated with blue bars whose heights are proportional to the percentage of enhancement of the KMnO4 reactivity. (G) Scheme of the bottom strand sites of the pMV158 dso whose DMS or KMnO4 sensitivity is modified upon binding of RepB to the nic locus. Once RepB is bound to its high affinity target, the bind locus, the protein would bind to its lower affinity target in the nic locus, then rendering some sites highly sensitive to DMS (red ellipses) or to KMnO4 (blue ellipses). Whereas most of the sites that increase their KMnO4 sensitivity are within the nic locus, the most distorted site was found close to the DDR. The IR-I cruciform of the pMV158 dso is represented since binding of RepB to the nic locus promotes its extrusion.

Mentions: At higher RepB concentrations (protein to DNA molar ratio of 50:1 or higher), in addition to the footprints derived from the binding of RepB to the bind locus, DMS footprints consisting of hyperexposed bands were observed in the nic locus when analysing the methylation pattern of the bottom strand on supercoiled pMV158 DNA (Figure 7A and B). The relative intensity of the hyperexposed bands augmented as the protein concentration was increased, until a plateau was reached at a protein to DNA molar ratio of 150:1 (not shown). The hypermethylated residues (mostly Gs) lie on the right arm of IR-I and on the adjacent DNA region which is complementary to the nick sequence (Figure 7G) and may reflect the proximity of RepB and/or changes in the DNA conformation due to extrusion of the corresponding cruciform. As a control, DMS footprinting analysis was also performed on supercoiled DNA of plasmid pCGA7, a derivative of pC194 which bears the separate pMV158-nic locus (including IR-I and the PDR) (12,15). The presence of RepB also resulted in hypermethylation of the same nucleotides as in pMV158, although it was evident that much higher concentrations of the protein were required to obtain similar levels of enhancement of the DMS reactivity in pCGA7 (Figure 7A and B), with no plateau being reached at the RepB concentrations assayed. These results suggested that binding of RepB to its secondary site in the nic locus might be facilitated in supercoiled DNAs containing the entire dso.Figure 7.


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)

DMS and KMnO4 footprints of RepB bound to the nic locus on supercoiled DNAs. (A) Sequencing gels displaying DMS sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, pCGA7, a pC194 derivative which bears the nic region of the pMV158 dso was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described under ‘Materials and Methods’. RepB concentrations were 0.36, 0.6, 1.2 and 1.8 µM (lanes 1–4) for the reactions containing pMV158 DNA, and 0.6, 1.2, 1.8 and 4.8 M (lanes 5–8) for the pCGA7 DNA. The methylation sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of IR-I, with its left (L) and right (R) arms and its central region harbouring the nick sequence, is shown. To the right, the DNA sequence of the bottom strand at the footprints is displayed with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are indicated with red boxes. The same bands in pCGA7 are in orange boxes. (B) Quantification of the experiment shown in A by PhosphorImager analysis using Quantity One software. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 of pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (red line for pMV158 and orange line for pCGA7) are superimposed. Bases whose DMS sensitivity is modified in the presence of RepB are indicated with red (pMV158) or orange (pCGA7) bars. Bar heights are proportional to the percentage of DMS sensitivity enhancement. (C) Sequencing gels displaying KMnO4 sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, plasmid pCGA7 was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described in ‘Materials and Methods’. RepB concentrations were 0.6 µM (lanes 1 and 5), 1.8 µM (lanes 2 and 6), 2.4 µM (lanes 3 and 7) and 4.8 µM (lanes 4 and 8). The modification sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of the central region and right arm (R) of IR-I is displayed. To the right, the DNA sequence of the bottom strand at the distorted DNA regions is shown with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are within blue boxes. The same bands in pCGA7 are within green boxes. The arrows point to sites of high RepB-independent KMnO4 reactivity. (D) Quantification of the experiment shown in C. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 corresponding to pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (blue line for pMV158 and green line for pCGA7) are superimposed. Bases whose KMnO4 sensitivity is modified in the presence of RepB are indicated with blue (pMV158) or green (pCGA7) bars. Bar heights are proportional to the percentage of enhancement in the KMnO4 reactivity. (E) Sequencing gels displaying KMnO4 sensitivity of the bottom strand close to the bind region, in the absence and in the presence of RepB. Supercoiled pMV158 DNA was incubated without RepB (–) or in the presence of 0.6, 1.8, 2.4 and 4.8 µM of the protein (lanes 1–4, respectively). KMnO4 reaction and mapping of the modification sites was as in C. A dideoxy sequencing ladder (lanes A, C, G, T) was included as a control. To the right, the DNA sequence of the bottom strand at the DNA region distorted upon binding of RepB to the nic locus is displayed with its 5′→3′ directionality. Bands whose intensity is modified in the presence of RepB are within a blue box. (F) Quantification of the experiment shown in ahown in E. Scans of lanes (–) (black line) and 4 (blue line) are drawn superimposed. Bases that increase their KMnO4 sensitivity in the presence of RepB are indicated with blue bars whose heights are proportional to the percentage of enhancement of the KMnO4 reactivity. (G) Scheme of the bottom strand sites of the pMV158 dso whose DMS or KMnO4 sensitivity is modified upon binding of RepB to the nic locus. Once RepB is bound to its high affinity target, the bind locus, the protein would bind to its lower affinity target in the nic locus, then rendering some sites highly sensitive to DMS (red ellipses) or to KMnO4 (blue ellipses). Whereas most of the sites that increase their KMnO4 sensitivity are within the nic locus, the most distorted site was found close to the DDR. The IR-I cruciform of the pMV158 dso is represented since binding of RepB to the nic locus promotes its extrusion.
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Figure 7: DMS and KMnO4 footprints of RepB bound to the nic locus on supercoiled DNAs. (A) Sequencing gels displaying DMS sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, pCGA7, a pC194 derivative which bears the nic region of the pMV158 dso was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described under ‘Materials and Methods’. RepB concentrations were 0.36, 0.6, 1.2 and 1.8 µM (lanes 1–4) for the reactions containing pMV158 DNA, and 0.6, 1.2, 1.8 and 4.8 M (lanes 5–8) for the pCGA7 DNA. The methylation sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of IR-I, with its left (L) and right (R) arms and its central region harbouring the nick sequence, is shown. To the right, the DNA sequence of the bottom strand at the footprints is displayed with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are indicated with red boxes. The same bands in pCGA7 are in orange boxes. (B) Quantification of the experiment shown in A by PhosphorImager analysis using Quantity One software. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 of pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (red line for pMV158 and orange line for pCGA7) are superimposed. Bases whose DMS sensitivity is modified in the presence of RepB are indicated with red (pMV158) or orange (pCGA7) bars. Bar heights are proportional to the percentage of DMS sensitivity enhancement. (C) Sequencing gels displaying KMnO4 sensitivity of the bottom strand around the nic region in the absence and in the presence of RepB. As a control, plasmid pCGA7 was used. Supercoiled pMV158 or pCGA7 DNAs were incubated in the absence of RepB (lanes –) or with various protein concentrations, and treated with DMS as described in ‘Materials and Methods’. RepB concentrations were 0.6 µM (lanes 1 and 5), 1.8 µM (lanes 2 and 6), 2.4 µM (lanes 3 and 7) and 4.8 µM (lanes 4 and 8). The modification sites on the bottom strand were mapped by primer extension using a labelled primer corresponding to the top strand. The same primer was used for the control dideoxy sequencing ladder (lanes A, C, G, T). On the left, the location of the central region and right arm (R) of IR-I is displayed. To the right, the DNA sequence of the bottom strand at the distorted DNA regions is shown with its 5′→3′ directionality. Bands whose intensity is modified in pMV158 in the presence of RepB are within blue boxes. The same bands in pCGA7 are within green boxes. The arrows point to sites of high RepB-independent KMnO4 reactivity. (D) Quantification of the experiment shown in C. Scans of lanes (–) and 4 corresponding to pMV158, and of lanes (–) and 8 corresponding to pCGA7 are drawn at the top and the bottom of the panel, respectively. The scans obtained with naked DNA (black line) and RepB-bound DNA (blue line for pMV158 and green line for pCGA7) are superimposed. Bases whose KMnO4 sensitivity is modified in the presence of RepB are indicated with blue (pMV158) or green (pCGA7) bars. Bar heights are proportional to the percentage of enhancement in the KMnO4 reactivity. (E) Sequencing gels displaying KMnO4 sensitivity of the bottom strand close to the bind region, in the absence and in the presence of RepB. Supercoiled pMV158 DNA was incubated without RepB (–) or in the presence of 0.6, 1.8, 2.4 and 4.8 µM of the protein (lanes 1–4, respectively). KMnO4 reaction and mapping of the modification sites was as in C. A dideoxy sequencing ladder (lanes A, C, G, T) was included as a control. To the right, the DNA sequence of the bottom strand at the DNA region distorted upon binding of RepB to the nic locus is displayed with its 5′→3′ directionality. Bands whose intensity is modified in the presence of RepB are within a blue box. (F) Quantification of the experiment shown in ahown in E. Scans of lanes (–) (black line) and 4 (blue line) are drawn superimposed. Bases that increase their KMnO4 sensitivity in the presence of RepB are indicated with blue bars whose heights are proportional to the percentage of enhancement of the KMnO4 reactivity. (G) Scheme of the bottom strand sites of the pMV158 dso whose DMS or KMnO4 sensitivity is modified upon binding of RepB to the nic locus. Once RepB is bound to its high affinity target, the bind locus, the protein would bind to its lower affinity target in the nic locus, then rendering some sites highly sensitive to DMS (red ellipses) or to KMnO4 (blue ellipses). Whereas most of the sites that increase their KMnO4 sensitivity are within the nic locus, the most distorted site was found close to the DDR. The IR-I cruciform of the pMV158 dso is represented since binding of RepB to the nic locus promotes its extrusion.
Mentions: At higher RepB concentrations (protein to DNA molar ratio of 50:1 or higher), in addition to the footprints derived from the binding of RepB to the bind locus, DMS footprints consisting of hyperexposed bands were observed in the nic locus when analysing the methylation pattern of the bottom strand on supercoiled pMV158 DNA (Figure 7A and B). The relative intensity of the hyperexposed bands augmented as the protein concentration was increased, until a plateau was reached at a protein to DNA molar ratio of 150:1 (not shown). The hypermethylated residues (mostly Gs) lie on the right arm of IR-I and on the adjacent DNA region which is complementary to the nick sequence (Figure 7G) and may reflect the proximity of RepB and/or changes in the DNA conformation due to extrusion of the corresponding cruciform. As a control, DMS footprinting analysis was also performed on supercoiled DNA of plasmid pCGA7, a derivative of pC194 which bears the separate pMV158-nic locus (including IR-I and the PDR) (12,15). The presence of RepB also resulted in hypermethylation of the same nucleotides as in pMV158, although it was evident that much higher concentrations of the protein were required to obtain similar levels of enhancement of the DMS reactivity in pCGA7 (Figure 7A and B), with no plateau being reached at the RepB concentrations assayed. These results suggested that binding of RepB to its secondary site in the nic locus might be facilitated in supercoiled DNAs containing the entire dso.Figure 7.

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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Related in: MedlinePlus