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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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Transformation efficiency of methylated oriI and oriC plasmids in E. coli.(A) The strains used were MG1655 and its dam16::km, ΔseqA10 and dam16::km ΔseqA10 derivatives, which are abbreviated as dam, seqA and dam seqA, respectively. The plasmids used carried the following origins: pBR322ori (in pBAD24; black bars), oriI (red bars), and oriC (orange bars). The transformation efficiency, expressed as colony forming units (CFU) per µg of DNA, is the average of three independent experiments. The error bars marked here and elsewhere represent one standard deviation of the mean. (B) The starting strain was MG1655ΔoriC::oriI, otherwise the details are as in (A).
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pgen-1000939-g001: Transformation efficiency of methylated oriI and oriC plasmids in E. coli.(A) The strains used were MG1655 and its dam16::km, ΔseqA10 and dam16::km ΔseqA10 derivatives, which are abbreviated as dam, seqA and dam seqA, respectively. The plasmids used carried the following origins: pBR322ori (in pBAD24; black bars), oriI (red bars), and oriC (orange bars). The transformation efficiency, expressed as colony forming units (CFU) per µg of DNA, is the average of three independent experiments. The error bars marked here and elsewhere represent one standard deviation of the mean. (B) The starting strain was MG1655ΔoriC::oriI, otherwise the details are as in (A).

Mentions: The E. coli origin of replication, oriC, does not require dam and seqA to initiate replication. In contrast, plasmids driven by oriC are highly deficient in transformation of dam mutants [11]. This is believed to be due to irreversible sequestration of hemimethylated plasmid origins by the SeqA protein after the first round of replication [22]. Indeed, seqA and dam seqA strains can be transformed by oriC plasmids, although the efficiency is lower compared to WT due to incompatibility with the chromosomal copy of the origin [15]. The requirement of dam thus is not intrinsic to oriC function and appears so only in the plasmid context. The dam requirement of V. cholerae oriI has so far been studied only in the plasmid context. However, in contrast to oriC plasmids, oriI plasmids not only failed to transform an E. coli dam mutant but also a seqA or a dam seqA mutant, raising the possibility that the genes could be essential for oriI [11], [18]. We confirmed the plasmid results using E. coli MG1655 (BR1703) and its dam (CVC1415), seqA (BR1704) and dam seqA (CVC1424) mutant derivatives. As before, the dam, seqA and dam seqA mutants could not be transformed with an oriI plasmid, and only the dam mutant could not be transformed with the oriC plasmid (Figure 1A). We suggest below that the oriI plasmid possibly replicated in the absence of dam or seqA, which competed out replication from the chromosomal oriC and led to inviability of the transformants.


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

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

Transformation efficiency of methylated oriI and oriC plasmids in E. coli.(A) The strains used were MG1655 and its dam16::km, ΔseqA10 and dam16::km ΔseqA10 derivatives, which are abbreviated as dam, seqA and dam seqA, respectively. The plasmids used carried the following origins: pBR322ori (in pBAD24; black bars), oriI (red bars), and oriC (orange bars). The transformation efficiency, expressed as colony forming units (CFU) per µg of DNA, is the average of three independent experiments. The error bars marked here and elsewhere represent one standard deviation of the mean. (B) The starting strain was MG1655ΔoriC::oriI, otherwise the details are as in (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865523&req=5

pgen-1000939-g001: Transformation efficiency of methylated oriI and oriC plasmids in E. coli.(A) The strains used were MG1655 and its dam16::km, ΔseqA10 and dam16::km ΔseqA10 derivatives, which are abbreviated as dam, seqA and dam seqA, respectively. The plasmids used carried the following origins: pBR322ori (in pBAD24; black bars), oriI (red bars), and oriC (orange bars). The transformation efficiency, expressed as colony forming units (CFU) per µg of DNA, is the average of three independent experiments. The error bars marked here and elsewhere represent one standard deviation of the mean. (B) The starting strain was MG1655ΔoriC::oriI, otherwise the details are as in (A).
Mentions: The E. coli origin of replication, oriC, does not require dam and seqA to initiate replication. In contrast, plasmids driven by oriC are highly deficient in transformation of dam mutants [11]. This is believed to be due to irreversible sequestration of hemimethylated plasmid origins by the SeqA protein after the first round of replication [22]. Indeed, seqA and dam seqA strains can be transformed by oriC plasmids, although the efficiency is lower compared to WT due to incompatibility with the chromosomal copy of the origin [15]. The requirement of dam thus is not intrinsic to oriC function and appears so only in the plasmid context. The dam requirement of V. cholerae oriI has so far been studied only in the plasmid context. However, in contrast to oriC plasmids, oriI plasmids not only failed to transform an E. coli dam mutant but also a seqA or a dam seqA mutant, raising the possibility that the genes could be essential for oriI [11], [18]. We confirmed the plasmid results using E. coli MG1655 (BR1703) and its dam (CVC1415), seqA (BR1704) and dam seqA (CVC1424) mutant derivatives. As before, the dam, seqA and dam seqA mutants could not be transformed with an oriI plasmid, and only the dam mutant could not be transformed with the oriC plasmid (Figure 1A). We suggest below that the oriI plasmid possibly replicated in the absence of dam or seqA, which competed out replication from the chromosomal oriC and led to inviability of the transformants.

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