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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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RepB-induced DNA bending characterized by circular permutation assays. Both 241-bp nic- (A) and 243-bp bind- (B) permuted fragments were generated by restriction with the enzymes indicated in the scheme. Below, the distance (d, in bp) from the centre of the cloned pMV158 DNA (coordinate in brackets) to the right end of each fragment is displayed. The electrophoretic mobility of free (closed circles) or bound (open circles) DNA fragments, normalized to the mobility of the slowest same-sized DNA or complex, was plotted versus d. The apparent angles of both the intrinsic curvature and the RepB-induced bending on the nic and bind loci are shown in Table 1.
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Figure 5: RepB-induced DNA bending characterized by circular permutation assays. Both 241-bp nic- (A) and 243-bp bind- (B) permuted fragments were generated by restriction with the enzymes indicated in the scheme. Below, the distance (d, in bp) from the centre of the cloned pMV158 DNA (coordinate in brackets) to the right end of each fragment is displayed. The electrophoretic mobility of free (closed circles) or bound (open circles) DNA fragments, normalized to the mobility of the slowest same-sized DNA or complex, was plotted versus d. The apparent angles of both the intrinsic curvature and the RepB-induced bending on the nic and bind loci are shown in Table 1.

Mentions: To explore the possible RepB-induced bending, we cloned separately the nic and bind loci of the pMV158-dso into the test vector pBEND2 to conduct circular permutation assays (24). This technique uses DNA bending constructs which can be cleaved at different restriction sites, generating DNA fragments of uniform length but with the suspected bend site placed at different positions with respect to the ends of the fragments (33). If a protein induces a bend, it will affect the relative electrophoretic mobility of the fragments in a predictable manner that allows calculation of the bend angles (34). The DNA fragments obtained by restriction with different enzymes were incubated with RepB to perform EMSA under conditions in which 50% of the DNA was bound in the corresponding C1 complex, so that in the same assay we could calculate the electrophoretic mobility of the free and bound DNAs. To calculate the relative mobilities, the ratio of the mobility of the bound to the unbound DNA with the binding site in the middle of the fragment (µMB/µM) was divided by the same ratio for the DNA with the binding site at the end of the fragment (µEB/µE). This method cancels out the contribution of the intrinsic curvature of the DNA, although it only gives accurate estimations when the induced bend is large compared with the intrinsic curvature (26). In addition, the bends induced by RepB on the nic and bind loci, although probably located at the same place as the intrinsic curvatures of the corresponding fragments (plots in Figure 5), might not coincide with them in the directionality. This could complicate the deconvolution of the effects of the intrinsic and induced bends. For these reasons, we also estimated the intrinsic curvature of the unbound DNAs (from µM/µE), as well as the total bending angle of the RepB-complexed DNAs (from µMB/µEB). The results (Table 1) demonstrated that the DNA of the bind locus exhibited an intrinsic bend of about 50°, whereas the nic locus was essentially straight. RepB induced a bend of 120° (105° when the intrinsic curvature of the DNA was deducted) on the DDR-containing DNA, whereas an apparent bending angle of around 60° was measured as induced by the protein on the fragment harbouring the nic region. Since the method used underestimates bending angles sharper than 100° (26), we conclude that RepB induces a very strong bend (>100°) on the bind locus, while binding of the protein to the nic locus results in a moderate bending of the DNA.Figure 5.


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)

RepB-induced DNA bending characterized by circular permutation assays. Both 241-bp nic- (A) and 243-bp bind- (B) permuted fragments were generated by restriction with the enzymes indicated in the scheme. Below, the distance (d, in bp) from the centre of the cloned pMV158 DNA (coordinate in brackets) to the right end of each fragment is displayed. The electrophoretic mobility of free (closed circles) or bound (open circles) DNA fragments, normalized to the mobility of the slowest same-sized DNA or complex, was plotted versus d. The apparent angles of both the intrinsic curvature and the RepB-induced bending on the nic and bind loci are shown in Table 1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: RepB-induced DNA bending characterized by circular permutation assays. Both 241-bp nic- (A) and 243-bp bind- (B) permuted fragments were generated by restriction with the enzymes indicated in the scheme. Below, the distance (d, in bp) from the centre of the cloned pMV158 DNA (coordinate in brackets) to the right end of each fragment is displayed. The electrophoretic mobility of free (closed circles) or bound (open circles) DNA fragments, normalized to the mobility of the slowest same-sized DNA or complex, was plotted versus d. The apparent angles of both the intrinsic curvature and the RepB-induced bending on the nic and bind loci are shown in Table 1.
Mentions: To explore the possible RepB-induced bending, we cloned separately the nic and bind loci of the pMV158-dso into the test vector pBEND2 to conduct circular permutation assays (24). This technique uses DNA bending constructs which can be cleaved at different restriction sites, generating DNA fragments of uniform length but with the suspected bend site placed at different positions with respect to the ends of the fragments (33). If a protein induces a bend, it will affect the relative electrophoretic mobility of the fragments in a predictable manner that allows calculation of the bend angles (34). The DNA fragments obtained by restriction with different enzymes were incubated with RepB to perform EMSA under conditions in which 50% of the DNA was bound in the corresponding C1 complex, so that in the same assay we could calculate the electrophoretic mobility of the free and bound DNAs. To calculate the relative mobilities, the ratio of the mobility of the bound to the unbound DNA with the binding site in the middle of the fragment (µMB/µM) was divided by the same ratio for the DNA with the binding site at the end of the fragment (µEB/µE). This method cancels out the contribution of the intrinsic curvature of the DNA, although it only gives accurate estimations when the induced bend is large compared with the intrinsic curvature (26). In addition, the bends induced by RepB on the nic and bind loci, although probably located at the same place as the intrinsic curvatures of the corresponding fragments (plots in Figure 5), might not coincide with them in the directionality. This could complicate the deconvolution of the effects of the intrinsic and induced bends. For these reasons, we also estimated the intrinsic curvature of the unbound DNAs (from µM/µE), as well as the total bending angle of the RepB-complexed DNAs (from µMB/µEB). The results (Table 1) demonstrated that the DNA of the bind locus exhibited an intrinsic bend of about 50°, whereas the nic locus was essentially straight. RepB induced a bend of 120° (105° when the intrinsic curvature of the DNA was deducted) on the DDR-containing DNA, whereas an apparent bending angle of around 60° was measured as induced by the protein on the fragment harbouring the nic region. Since the method used underestimates bending angles sharper than 100° (26), we conclude that RepB induces a very strong bend (>100°) on the bind locus, while binding of the protein to the nic locus results in a moderate bending of the DNA.Figure 5.

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