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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

Handcuffing of three 11-mer carrying fragments by RctB. A fragment with three tandem 11-mers of incII (Figure 1) was obtained from pTVC248 and reacted with varying RctB concentrations. The proteins tested were WT, ΔC157 and F378S. One half of the binding mixture was loaded onto 5% polyacrylamide gel to monitor bound DNA by EMSA (bracket B) (top panel). The position of the free probe is marked shown by ‘F’. The other half of the binding mixture was treated with ligase, and after deproteinization, loaded onto 1.2% agarose gel to monitor ladder (L) and monomer circle (C) formation (middle panel). The bottom panel represents graphically the efficiency of ladder formation for the WT and the two mutants. Percentage of ladder for each lane was calculated by taking intensity of the area (marked L) containing all bands above free (F) DNA and monomer circles (C), after subtracting the intensity of the similar area in lane 1 (without RctB) and further dividing by the total intensity of bands.
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gks260-F7: Handcuffing of three 11-mer carrying fragments by RctB. A fragment with three tandem 11-mers of incII (Figure 1) was obtained from pTVC248 and reacted with varying RctB concentrations. The proteins tested were WT, ΔC157 and F378S. One half of the binding mixture was loaded onto 5% polyacrylamide gel to monitor bound DNA by EMSA (bracket B) (top panel). The position of the free probe is marked shown by ‘F’. The other half of the binding mixture was treated with ligase, and after deproteinization, loaded onto 1.2% agarose gel to monitor ladder (L) and monomer circle (C) formation (middle panel). The bottom panel represents graphically the efficiency of ladder formation for the WT and the two mutants. Percentage of ladder for each lane was calculated by taking intensity of the area (marked L) containing all bands above free (F) DNA and monomer circles (C), after subtracting the intensity of the similar area in lane 1 (without RctB) and further dividing by the total intensity of bands.

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)

Handcuffing of three 11-mer carrying fragments by RctB. A fragment with three tandem 11-mers of incII (Figure 1) was obtained from pTVC248 and reacted with varying RctB concentrations. The proteins tested were WT, ΔC157 and F378S. One half of the binding mixture was loaded onto 5% polyacrylamide gel to monitor bound DNA by EMSA (bracket B) (top panel). The position of the free probe is marked shown by ‘F’. The other half of the binding mixture was treated with ligase, and after deproteinization, loaded onto 1.2% agarose gel to monitor ladder (L) and monomer circle (C) formation (middle panel). The bottom panel represents graphically the efficiency of ladder formation for the WT and the two mutants. Percentage of ladder for each lane was calculated by taking intensity of the area (marked L) containing all bands above free (F) DNA and monomer circles (C), after subtracting the intensity of the similar area in lane 1 (without RctB) and further dividing by the total intensity of bands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks260-F7: Handcuffing of three 11-mer carrying fragments by RctB. A fragment with three tandem 11-mers of incII (Figure 1) was obtained from pTVC248 and reacted with varying RctB concentrations. The proteins tested were WT, ΔC157 and F378S. One half of the binding mixture was loaded onto 5% polyacrylamide gel to monitor bound DNA by EMSA (bracket B) (top panel). The position of the free probe is marked shown by ‘F’. The other half of the binding mixture was treated with ligase, and after deproteinization, loaded onto 1.2% agarose gel to monitor ladder (L) and monomer circle (C) formation (middle panel). The bottom panel represents graphically the efficiency of ladder formation for the WT and the two mutants. Percentage of ladder for each lane was calculated by taking intensity of the area (marked L) containing all bands above free (F) DNA and monomer circles (C), after subtracting the intensity of the similar area in lane 1 (without RctB) and further dividing by the total intensity of bands.
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