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Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer.

Jha JK, Demarre G, Venkova-Canova T, Chattoraj DK - Nucleic Acids Res. (2012)

Bottom Line: Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators.ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo.The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, NCI, 37 Convent Drive, NIH, Bethesda, MD 20892-4260, USA.

ABSTRACT
The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.

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Related in: MedlinePlus

EMSA of RctB binding to fragments carrying a 12- or a 39-mer. EMSA was performed with end-labeled (32P) DNA fragments carrying a single copy of either a 12-mer or a 39-mer with 55 bp of vector sequences at both flanks (obtained from plasmids pTVC195 or pTVC174, respectively). RctB was either WT or mutants ΔC157 and F378S, and used in the concentration range of 0.67–33 nM. The binding was analyzed using a 5% polyacrlyamide gel. Arrows show two retarded bands of the 12-mer fragment. The binding profiles are shown at the bottom. The data points were fitted to equation , nH = Hill slope and Bmax = maximum %bound. The values of nH for the WT, ΔC157 and F378S were 1.8, 1.2 and 1.4 for the 12-mer, and 1.7, 1 and 5 for the 39-mer.
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gks260-F3: EMSA of RctB binding to fragments carrying a 12- or a 39-mer. EMSA was performed with end-labeled (32P) DNA fragments carrying a single copy of either a 12-mer or a 39-mer with 55 bp of vector sequences at both flanks (obtained from plasmids pTVC195 or pTVC174, respectively). RctB was either WT or mutants ΔC157 and F378S, and used in the concentration range of 0.67–33 nM. The binding was analyzed using a 5% polyacrlyamide gel. Arrows show two retarded bands of the 12-mer fragment. The binding profiles are shown at the bottom. The data points were fitted to equation , nH = Hill slope and Bmax = maximum %bound. The values of nH for the WT, ΔC157 and F378S were 1.8, 1.2 and 1.4 for the 12-mer, and 1.7, 1 and 5 for the 39-mer.

Mentions: Bacterial strains and plasmids used in this study


Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer.

Jha JK, Demarre G, Venkova-Canova T, Chattoraj DK - Nucleic Acids Res. (2012)

EMSA of RctB binding to fragments carrying a 12- or a 39-mer. EMSA was performed with end-labeled (32P) DNA fragments carrying a single copy of either a 12-mer or a 39-mer with 55 bp of vector sequences at both flanks (obtained from plasmids pTVC195 or pTVC174, respectively). RctB was either WT or mutants ΔC157 and F378S, and used in the concentration range of 0.67–33 nM. The binding was analyzed using a 5% polyacrlyamide gel. Arrows show two retarded bands of the 12-mer fragment. The binding profiles are shown at the bottom. The data points were fitted to equation , nH = Hill slope and Bmax = maximum %bound. The values of nH for the WT, ΔC157 and F378S were 1.8, 1.2 and 1.4 for the 12-mer, and 1.7, 1 and 5 for the 39-mer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401445&req=5

gks260-F3: EMSA of RctB binding to fragments carrying a 12- or a 39-mer. EMSA was performed with end-labeled (32P) DNA fragments carrying a single copy of either a 12-mer or a 39-mer with 55 bp of vector sequences at both flanks (obtained from plasmids pTVC195 or pTVC174, respectively). RctB was either WT or mutants ΔC157 and F378S, and used in the concentration range of 0.67–33 nM. The binding was analyzed using a 5% polyacrlyamide gel. Arrows show two retarded bands of the 12-mer fragment. The binding profiles are shown at the bottom. The data points were fitted to equation , nH = Hill slope and Bmax = maximum %bound. The values of nH for the WT, ΔC157 and F378S were 1.8, 1.2 and 1.4 for the 12-mer, and 1.7, 1 and 5 for the 39-mer.
Mentions: Bacterial strains and plasmids used in this study

Bottom Line: Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators.ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo.The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, NCI, 37 Convent Drive, NIH, Bethesda, MD 20892-4260, USA.

ABSTRACT
The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.

Show MeSH
Related in: MedlinePlus