E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration

@article{Slater1995ECS,
  title={E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration},
  author={S. Slater and S. Wold and M. Lu and E. Boye and K. Skarstad and N. Kleckner},
  journal={Cell},
  year={1995},
  volume={82},
  pages={927-936}
}
The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully… Expand
SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor
TLDR
It is demonstrated that SeqA stimulates transcription from the bacteriophage λpR promoter both in vivo and in vitro, and is concluded that, apart from its function in the control of DNA replication, SequA is also a specific transcription factor. Expand
The Escherichia coli SeqA protein binds specifically and co‐operatively to two sites in hemimethylated and fully methylated oriC
TLDR
It is suggested that SeqA binds to two nucleation sites in oriC, and by the aid of protein–protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates. Expand
The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology.
TLDR
In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU. Expand
SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC.
TLDR
This paper reports that oriC resets to ORC during sequestration, which was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-Affinity sites. Expand
Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC
TLDR
SeqA restrained the negative supercoils of unmethylated oriC plasmids, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC, and seemed to act more cooperatively than the effect of HU on plasmid topology. Expand
SeqA, the Escherichia coli origin sequestration protein, can regulate the replication of the pBR322 plasmid.
TLDR
It is suggested that the SeqA protein could modulate periodically the initiation of replication of the pBR322 plasmid, and could be fixed by its origin sequence, on a hemimethylated state, during the Initiation of the replication. Expand
Effects of purified SeqA protein on oriC‐dependent DNA replication in vitro
TLDR
The data suggest that SeqA participates in the assembly of initiation‐competent complexes at oriC and, at a later stage, influences the behaviour of these complexes, and alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations ofDnaA. Expand
Dynamic Distribution of SeqA Protein across the Chromosome of Escherichia coli K-12
TLDR
A study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays, suggests that sequential changes in SequA distribution orchestrate a program of gene expression that ensures coordinated DNA replication and cell division. Expand
Stimulation of the lambda pR promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation.
TLDR
In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p(R) transcription occurs after open complex formation, namely during promoter clearance, which is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter. Expand
Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli
TLDR
Results indicate that sequestration A binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated, which supports the translocating replication apparatuses model. Expand
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References

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A protein that binds to the P1 origin core and the oriC 13mer region in a methylation‐specific fashion is the product of the host seqA gene.
TLDR
The role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding and it is concluded that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. Expand
E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork
TLDR
Evidence is presented that oriC is a single function unit that is specifically sequestered from dam methyltransferase for a significant period of time and then released, and that the dnaA promoter region is subject to sequestration analogous to that observed at oriC and thus that hemimethylation-dependent sequestration is a general phenomenon. Expand
SeqA: A negative modulator of replication initiation in E. coli
TLDR
The identification of a gene required for sequestration is reported and it is demonstrated that this gene, seqA, also serves as a negative modulator of the primary initiation process, suggesting that SeqA might be a cooperativity factor, acting to make the replication initiation process dependent upon cooperative interactions among components. Expand
Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA–DNA hybrid.
TLDR
Activation of oriC by the distantly located R‐loop appears to require propagation of DNA melting through the intervening sequence. Expand
Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.
TLDR
In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis, suggesting a new function for the Dam methylase. Expand
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TLDR
It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E. coli cells with the polA1hip3 double mutation and it is proposed that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required. Expand
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TLDR
A footprinting analysis with the outer membrane of Escherichia coli demonstrates that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Expand
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TLDR
It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin, and it is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time needed to terminate mIOC transcription. Expand
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TLDR
First in vivo evidence for a structural change at the 13mers during initiation complex formation is observed at oriC, the leftmost region of the Escherichia coli origin of DNA replication. Expand
The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli.
TLDR
Direct evidence is provided that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box and are known to destabilize the tightly bound ATP. Expand
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