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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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Quantification of hemimethylated GATC sites in oriC and oriI in dam and seqA mutants of E. coli.(A) Schematic maps of origin regions of E. coli chromosome and V. cholerae chromosome I. The regions are very similar except that the V. cholerae origin (oriI) has an extra DnaA box (R6). Other details are described in Figure 5A. The tau and I sites of the E. coli origin (oriC) that also bind DnaA are yet to be described in oriI. The two GATC sites studied here for their methylation states are shown by vertical arrows. (B) The WT either had oriC (MG1655) or oriI (MG1655ΔoriC::oriI). The GATC sites probed were located either within the origins (oriC or oriI) or external to the origin (ext) at about 300 kb away. The same ext marker was used for both the strains. The numbers below the figure show the percent of hemimethylated DNA at the origins. N.A. stands for ‘not applicable’; in these lanes the DNA being from a dam mutant is unmethylated, and is all cleaved both at the origin and the external markers. An uncharacterized cross-reacting band appears in the dam mutants only (arrow head).
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pgen-1000939-g007: Quantification of hemimethylated GATC sites in oriC and oriI in dam and seqA mutants of E. coli.(A) Schematic maps of origin regions of E. coli chromosome and V. cholerae chromosome I. The regions are very similar except that the V. cholerae origin (oriI) has an extra DnaA box (R6). Other details are described in Figure 5A. The tau and I sites of the E. coli origin (oriC) that also bind DnaA are yet to be described in oriI. The two GATC sites studied here for their methylation states are shown by vertical arrows. (B) The WT either had oriC (MG1655) or oriI (MG1655ΔoriC::oriI). The GATC sites probed were located either within the origins (oriC or oriI) or external to the origin (ext) at about 300 kb away. The same ext marker was used for both the strains. The numbers below the figure show the percent of hemimethylated DNA at the origins. N.A. stands for ‘not applicable’; in these lanes the DNA being from a dam mutant is unmethylated, and is all cleaved both at the origin and the external markers. An uncharacterized cross-reacting band appears in the dam mutants only (arrow head).

Mentions: The opposite response of the GATC sites tested in oriI and oriII was also seen in a V. cholerae mutant where seqA was completely deleted (ΔseqAT, CVC2003; Figure S4). oriI also responded opposite to oriC in E. coli (Figure 7). While the percent of hemimethylated DNA at oriC dropped from 13% in MG1655 to 9% in MG1655ΔseqA10, the values at oriI increased from 9% in MG1655ΔoriC::oriI to 25% in its ΔseqA10 derivative. These results suggest that the opposite behavior of oriI and oriC upon seqA deletion is intrinsic to the sequence context of the GATC sites tested in the two origins rather than the sequestration machinery of the two bacteria. Thus depending upon the context, SeqA can both shorten and prolong the hemimethylation period of a GATC site.


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

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

Quantification of hemimethylated GATC sites in oriC and oriI in dam and seqA mutants of E. coli.(A) Schematic maps of origin regions of E. coli chromosome and V. cholerae chromosome I. The regions are very similar except that the V. cholerae origin (oriI) has an extra DnaA box (R6). Other details are described in Figure 5A. The tau and I sites of the E. coli origin (oriC) that also bind DnaA are yet to be described in oriI. The two GATC sites studied here for their methylation states are shown by vertical arrows. (B) The WT either had oriC (MG1655) or oriI (MG1655ΔoriC::oriI). The GATC sites probed were located either within the origins (oriC or oriI) or external to the origin (ext) at about 300 kb away. The same ext marker was used for both the strains. The numbers below the figure show the percent of hemimethylated DNA at the origins. N.A. stands for ‘not applicable’; in these lanes the DNA being from a dam mutant is unmethylated, and is all cleaved both at the origin and the external markers. An uncharacterized cross-reacting band appears in the dam mutants only (arrow head).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865523&req=5

pgen-1000939-g007: Quantification of hemimethylated GATC sites in oriC and oriI in dam and seqA mutants of E. coli.(A) Schematic maps of origin regions of E. coli chromosome and V. cholerae chromosome I. The regions are very similar except that the V. cholerae origin (oriI) has an extra DnaA box (R6). Other details are described in Figure 5A. The tau and I sites of the E. coli origin (oriC) that also bind DnaA are yet to be described in oriI. The two GATC sites studied here for their methylation states are shown by vertical arrows. (B) The WT either had oriC (MG1655) or oriI (MG1655ΔoriC::oriI). The GATC sites probed were located either within the origins (oriC or oriI) or external to the origin (ext) at about 300 kb away. The same ext marker was used for both the strains. The numbers below the figure show the percent of hemimethylated DNA at the origins. N.A. stands for ‘not applicable’; in these lanes the DNA being from a dam mutant is unmethylated, and is all cleaved both at the origin and the external markers. An uncharacterized cross-reacting band appears in the dam mutants only (arrow head).
Mentions: The opposite response of the GATC sites tested in oriI and oriII was also seen in a V. cholerae mutant where seqA was completely deleted (ΔseqAT, CVC2003; Figure S4). oriI also responded opposite to oriC in E. coli (Figure 7). While the percent of hemimethylated DNA at oriC dropped from 13% in MG1655 to 9% in MG1655ΔseqA10, the values at oriI increased from 9% in MG1655ΔoriC::oriI to 25% in its ΔseqA10 derivative. These results suggest that the opposite behavior of oriI and oriC upon seqA deletion is intrinsic to the sequence context of the GATC sites tested in the two origins rather than the sequestration machinery of the two bacteria. Thus depending upon the context, SeqA can both shorten and prolong the hemimethylation period of a GATC site.

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