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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

Flow cytometric analysis of DNA content in E. coli.The cells used were MG1655 (A) and MG1655ΔoriC::oriI (B), and their dam and seqA mutant derivatives (C, E) and (D, F), respectively. Cells were analyzed after replication-run out in the presence of drugs that inhibit replication initiation and cell division. 100,000 cells were analyzed in each experiment.
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pgen-1000939-g002: Flow cytometric analysis of DNA content in E. coli.The cells used were MG1655 (A) and MG1655ΔoriC::oriI (B), and their dam and seqA mutant derivatives (C, E) and (D, F), respectively. Cells were analyzed after replication-run out in the presence of drugs that inhibit replication initiation and cell division. 100,000 cells were analyzed in each experiment.

Mentions: oriI and oriC were further analyzed using flow cytometry [25]. Replication initiation and cell division were blocked by antibiotics rifampicin and cephalexin, respectively, but sufficient time was allowed after drug addition to complete replication elongation (replication run-out). This method provides a measure of the fraction of the population that already initiated replication at the time of drug addition. In LB, after the replication run-out, MG1655 cells were distributed mostly into two populations, one with four and the other with eight full chromosomes (Figure 2A). This indicates that cells were born with four origins and they all fired synchronously once, giving rise to the eight chromosome peak. In the dam and seqA mutants, cells had a widely varying number of chromosomes indicating asynchronous initiation (Figure 2C and 2E) [22], [26]. There were also cells with more than eight chromosomes indicating that initiation was no longer restricted to once per cell cycle. In the engineered strain, MG1655ΔoriC::oriI, replication initiation was synchronous (Figure 2B) but not in its dam or seqA derivatives (Figure 2D and 2F). The requirements of dam and seqA for synchronous and once-per-cell-cycle initiation are thus maintained when oriI replaces oriC.


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

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

Flow cytometric analysis of DNA content in E. coli.The cells used were MG1655 (A) and MG1655ΔoriC::oriI (B), and their dam and seqA mutant derivatives (C, E) and (D, F), respectively. Cells were analyzed after replication-run out in the presence of drugs that inhibit replication initiation and cell division. 100,000 cells were analyzed in each experiment.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000939-g002: Flow cytometric analysis of DNA content in E. coli.The cells used were MG1655 (A) and MG1655ΔoriC::oriI (B), and their dam and seqA mutant derivatives (C, E) and (D, F), respectively. Cells were analyzed after replication-run out in the presence of drugs that inhibit replication initiation and cell division. 100,000 cells were analyzed in each experiment.
Mentions: oriI and oriC were further analyzed using flow cytometry [25]. Replication initiation and cell division were blocked by antibiotics rifampicin and cephalexin, respectively, but sufficient time was allowed after drug addition to complete replication elongation (replication run-out). This method provides a measure of the fraction of the population that already initiated replication at the time of drug addition. In LB, after the replication run-out, MG1655 cells were distributed mostly into two populations, one with four and the other with eight full chromosomes (Figure 2A). This indicates that cells were born with four origins and they all fired synchronously once, giving rise to the eight chromosome peak. In the dam and seqA mutants, cells had a widely varying number of chromosomes indicating asynchronous initiation (Figure 2C and 2E) [22], [26]. There were also cells with more than eight chromosomes indicating that initiation was no longer restricted to once per cell cycle. In the engineered strain, MG1655ΔoriC::oriI, replication initiation was synchronous (Figure 2B) but not in its dam or seqA derivatives (Figure 2D and 2F). The requirements of dam and seqA for synchronous and once-per-cell-cycle initiation are thus maintained when oriI replaces oriC.

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