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DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

Demarre G, Chattoraj DK - PLoS Genet. (2010)

Bottom Line: We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding.The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae.The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

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Related in: MedlinePlus

Localization of oriI and oriII in WT and ΔseqAP strains of V. cholerae.oriI and oriII were localized in exponentially growing cells of WT V. cholerae: CVC769 for oriI (A) and CVC827 for oriII (C), and the seqAP mutant: CVC1457 for oriI (B) and CVC1455 for oriII (D). The localization was done using the GFP–P1ParB/parS system. oriI was marked by inserting P1parS at about 90 kb away (counterclockwise to the origin), and oriII by inserting P1parS at about 40 kb away (clockwise to the origin). Plots show focus positions in cells with one (blue) focus, and two (red), three (light green), four (purple), five (orange), six (dark green), seven (pink), eight (brown) and nine (gray) foci. Focus positions were measured from a pole from which the distance to the nearest focus was smaller, and these (proximal) poles were placed on the abscissa. The other (distal) pole is shown as black circles. 200 cells were analyzed in each experiment. Only cells shorter than 6.25 µm were plotted in all cases. Longer cells accounted for 0 and 0.4% of total cells in WT (A, C), and 4.5 and 6% in the seqA mutant (B, D). (E) Distribution of cells vs. the number of foci they contained.
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pgen-1000939-g008: Localization of oriI and oriII in WT and ΔseqAP strains of V. cholerae.oriI and oriII were localized in exponentially growing cells of WT V. cholerae: CVC769 for oriI (A) and CVC827 for oriII (C), and the seqAP mutant: CVC1457 for oriI (B) and CVC1455 for oriII (D). The localization was done using the GFP–P1ParB/parS system. oriI was marked by inserting P1parS at about 90 kb away (counterclockwise to the origin), and oriII by inserting P1parS at about 40 kb away (clockwise to the origin). Plots show focus positions in cells with one (blue) focus, and two (red), three (light green), four (purple), five (orange), six (dark green), seven (pink), eight (brown) and nine (gray) foci. Focus positions were measured from a pole from which the distance to the nearest focus was smaller, and these (proximal) poles were placed on the abscissa. The other (distal) pole is shown as black circles. 200 cells were analyzed in each experiment. Only cells shorter than 6.25 µm were plotted in all cases. Longer cells accounted for 0 and 0.4% of total cells in WT (A, C), and 4.5 and 6% in the seqA mutant (B, D). (E) Distribution of cells vs. the number of foci they contained.

Mentions: Although a role of SeqA in restraining replication initiation in V. cholerae was suggested by the flow cytometry results (Figure S2B), they did not allow us to distinguish whether one or both the chromosomes were affected. We used fluorescence microscopy to follow replication initiation of the two chromosomes individually. The numbers and positions of oriI and oriII were determined in WT and ΔseqAP strains of V. cholerae by the GFP-P1ParB/parS system [30], [31]. For oriI in WT, 94% of the cells had two to four foci and the rest one or three foci, indicating synchronous and once-per-cell-cycle initiation (Figure 8A and 8E). In contrast, only 45% of ΔseqAP cells showed this pattern (Figure 8B and 8E). The remaining cells had five to nine foci. The significant increase in the number of cells with odd numbers of foci and more than four foci indicates that initiation is no longer synchronous and no longer limited to once per cell cycle in the absence of SeqA.


DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

Demarre G, Chattoraj DK - PLoS Genet. (2010)

Localization of oriI and oriII in WT and ΔseqAP strains of V. cholerae.oriI and oriII were localized in exponentially growing cells of WT V. cholerae: CVC769 for oriI (A) and CVC827 for oriII (C), and the seqAP mutant: CVC1457 for oriI (B) and CVC1455 for oriII (D). The localization was done using the GFP–P1ParB/parS system. oriI was marked by inserting P1parS at about 90 kb away (counterclockwise to the origin), and oriII by inserting P1parS at about 40 kb away (clockwise to the origin). Plots show focus positions in cells with one (blue) focus, and two (red), three (light green), four (purple), five (orange), six (dark green), seven (pink), eight (brown) and nine (gray) foci. Focus positions were measured from a pole from which the distance to the nearest focus was smaller, and these (proximal) poles were placed on the abscissa. The other (distal) pole is shown as black circles. 200 cells were analyzed in each experiment. Only cells shorter than 6.25 µm were plotted in all cases. Longer cells accounted for 0 and 0.4% of total cells in WT (A, C), and 4.5 and 6% in the seqA mutant (B, D). (E) Distribution of cells vs. the number of foci they contained.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865523&req=5

pgen-1000939-g008: Localization of oriI and oriII in WT and ΔseqAP strains of V. cholerae.oriI and oriII were localized in exponentially growing cells of WT V. cholerae: CVC769 for oriI (A) and CVC827 for oriII (C), and the seqAP mutant: CVC1457 for oriI (B) and CVC1455 for oriII (D). The localization was done using the GFP–P1ParB/parS system. oriI was marked by inserting P1parS at about 90 kb away (counterclockwise to the origin), and oriII by inserting P1parS at about 40 kb away (clockwise to the origin). Plots show focus positions in cells with one (blue) focus, and two (red), three (light green), four (purple), five (orange), six (dark green), seven (pink), eight (brown) and nine (gray) foci. Focus positions were measured from a pole from which the distance to the nearest focus was smaller, and these (proximal) poles were placed on the abscissa. The other (distal) pole is shown as black circles. 200 cells were analyzed in each experiment. Only cells shorter than 6.25 µm were plotted in all cases. Longer cells accounted for 0 and 0.4% of total cells in WT (A, C), and 4.5 and 6% in the seqA mutant (B, D). (E) Distribution of cells vs. the number of foci they contained.
Mentions: Although a role of SeqA in restraining replication initiation in V. cholerae was suggested by the flow cytometry results (Figure S2B), they did not allow us to distinguish whether one or both the chromosomes were affected. We used fluorescence microscopy to follow replication initiation of the two chromosomes individually. The numbers and positions of oriI and oriII were determined in WT and ΔseqAP strains of V. cholerae by the GFP-P1ParB/parS system [30], [31]. For oriI in WT, 94% of the cells had two to four foci and the rest one or three foci, indicating synchronous and once-per-cell-cycle initiation (Figure 8A and 8E). In contrast, only 45% of ΔseqAP cells showed this pattern (Figure 8B and 8E). The remaining cells had five to nine foci. The significant increase in the number of cells with odd numbers of foci and more than four foci indicates that initiation is no longer synchronous and no longer limited to once per cell cycle in the absence of SeqA.

Bottom Line: We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding.The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae.The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

Show MeSH
Related in: MedlinePlus