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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

Quantification of hemimethylated DNA in V. cholerae.(A) Schematic maps of origin regions of the two V. cholerae chromosomes. Both the chromosomal origins (oriI and oriII) are enriched in GATC sites (black dots). Their relative locations are shown with respect to some other features of the origins. For oriI, the features are the flanking genes gidA and mioC, an AT rich region, DnaA boxes (R1–R6) and an IHF site. The features for oriII are described in Figure 3A. Vertical arrows show the two GATC sites studied here for their methylation status. (B) Restriction enzyme names, recognition sequences and their cleavability before and after replication. The recognition sequences are shown in capital letters, and the remainder of the overlapping GATC site is shown in small letters. Prior to replication, these sites are fullymethylated (shown by the attached CH3 group on the adenine residues of both the strands) and are uncleavable (indicated by U); passage of the replication fork generates two hemimethylated products, one remains uncleavable but the other becomes cleavable (indicated by U and C, respectively). Thus, the percent of hemimethylated DNA is twice the percentage of cleavable DNA. (C) Probing of the hemimethylation state of GATC sites located either within the origins (oriI or oriII) or external to the origins (extI or extII) at about 300 kb away. Autoradiographs of Southern blots show sets of three lanes representing repeat experiments from independent cultures. (D) Quantification of band intensities. The bars represent the mean result of the set of three lanes. The experiments were done in LB (black bars) and in MM (M63 medium with casamino acids; gray bars).
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pgen-1000939-g005: Quantification of hemimethylated DNA in V. cholerae.(A) Schematic maps of origin regions of the two V. cholerae chromosomes. Both the chromosomal origins (oriI and oriII) are enriched in GATC sites (black dots). Their relative locations are shown with respect to some other features of the origins. For oriI, the features are the flanking genes gidA and mioC, an AT rich region, DnaA boxes (R1–R6) and an IHF site. The features for oriII are described in Figure 3A. Vertical arrows show the two GATC sites studied here for their methylation status. (B) Restriction enzyme names, recognition sequences and their cleavability before and after replication. The recognition sequences are shown in capital letters, and the remainder of the overlapping GATC site is shown in small letters. Prior to replication, these sites are fullymethylated (shown by the attached CH3 group on the adenine residues of both the strands) and are uncleavable (indicated by U); passage of the replication fork generates two hemimethylated products, one remains uncleavable but the other becomes cleavable (indicated by U and C, respectively). Thus, the percent of hemimethylated DNA is twice the percentage of cleavable DNA. (C) Probing of the hemimethylation state of GATC sites located either within the origins (oriI or oriII) or external to the origins (extI or extII) at about 300 kb away. Autoradiographs of Southern blots show sets of three lanes representing repeat experiments from independent cultures. (D) Quantification of band intensities. The bars represent the mean result of the set of three lanes. The experiments were done in LB (black bars) and in MM (M63 medium with casamino acids; gray bars).

Mentions: The hemimethylation period, the time to remethylate a GATC site after passage of the replication fork, is particularly prolonged at oriC because of the presence of high density of GATC sites within the origin [12]. The prevalence of high density of GATC sites in both oriI and oriII (Figure 5A) prompted us to examine their hemimethylation period, as was done using asynchronous exponential cultures [27], [28].


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 DNA in V. cholerae.(A) Schematic maps of origin regions of the two V. cholerae chromosomes. Both the chromosomal origins (oriI and oriII) are enriched in GATC sites (black dots). Their relative locations are shown with respect to some other features of the origins. For oriI, the features are the flanking genes gidA and mioC, an AT rich region, DnaA boxes (R1–R6) and an IHF site. The features for oriII are described in Figure 3A. Vertical arrows show the two GATC sites studied here for their methylation status. (B) Restriction enzyme names, recognition sequences and their cleavability before and after replication. The recognition sequences are shown in capital letters, and the remainder of the overlapping GATC site is shown in small letters. Prior to replication, these sites are fullymethylated (shown by the attached CH3 group on the adenine residues of both the strands) and are uncleavable (indicated by U); passage of the replication fork generates two hemimethylated products, one remains uncleavable but the other becomes cleavable (indicated by U and C, respectively). Thus, the percent of hemimethylated DNA is twice the percentage of cleavable DNA. (C) Probing of the hemimethylation state of GATC sites located either within the origins (oriI or oriII) or external to the origins (extI or extII) at about 300 kb away. Autoradiographs of Southern blots show sets of three lanes representing repeat experiments from independent cultures. (D) Quantification of band intensities. The bars represent the mean result of the set of three lanes. The experiments were done in LB (black bars) and in MM (M63 medium with casamino acids; gray bars).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865523&req=5

pgen-1000939-g005: Quantification of hemimethylated DNA in V. cholerae.(A) Schematic maps of origin regions of the two V. cholerae chromosomes. Both the chromosomal origins (oriI and oriII) are enriched in GATC sites (black dots). Their relative locations are shown with respect to some other features of the origins. For oriI, the features are the flanking genes gidA and mioC, an AT rich region, DnaA boxes (R1–R6) and an IHF site. The features for oriII are described in Figure 3A. Vertical arrows show the two GATC sites studied here for their methylation status. (B) Restriction enzyme names, recognition sequences and their cleavability before and after replication. The recognition sequences are shown in capital letters, and the remainder of the overlapping GATC site is shown in small letters. Prior to replication, these sites are fullymethylated (shown by the attached CH3 group on the adenine residues of both the strands) and are uncleavable (indicated by U); passage of the replication fork generates two hemimethylated products, one remains uncleavable but the other becomes cleavable (indicated by U and C, respectively). Thus, the percent of hemimethylated DNA is twice the percentage of cleavable DNA. (C) Probing of the hemimethylation state of GATC sites located either within the origins (oriI or oriII) or external to the origins (extI or extII) at about 300 kb away. Autoradiographs of Southern blots show sets of three lanes representing repeat experiments from independent cultures. (D) Quantification of band intensities. The bars represent the mean result of the set of three lanes. The experiments were done in LB (black bars) and in MM (M63 medium with casamino acids; gray bars).
Mentions: The hemimethylation period, the time to remethylate a GATC site after passage of the replication fork, is particularly prolonged at oriC because of the presence of high density of GATC sites within the origin [12]. The prevalence of high density of GATC sites in both oriI and oriII (Figure 5A) prompted us to examine their hemimethylation period, as was done using asynchronous exponential cultures [27], [28].

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