The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run‐out of replication in rapidly growing B/r A and K‐12 cultures, a small fraction of the cells (2‐7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.

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 methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

No abstract

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with Gl, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%o. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA(Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that forwild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.Normally, cells growing under steady-state conditions duplicate their chromosomal complement once and only once between each cell division. In any given steady-state culture of Escherichia coli cells, initiation of DNA replication at the chromosomal origin, oriC, occurs at a specific time in the cell cycle and at a specific cell mass. Two questions regarding the relationship between cell growth and initiation are of particular importance.First, how is the timing of initiation coupled to cell growth? An early hypothesis suggested that the initiation process responds to cell mass per se (1). According to this model, initiation always occurs at a fixed ratio of mass to origins-i.e., the initiation mass is a constant which is independent of the growth conditions. More recent evidence suggests, however, that the situation is more complicated, since the experimentally determined initiation mass varies significantly with growth rate (2, 3).Second, how variable is the mass at the time of initiation, when the cells grow under steady-state conditions? The extent of variation indicates the tightness of coupling between initiation and cell physiology, of which cell mass is an indicator: the less the variation, the tighter the coupling.Appropriately controlled replication initiation requires the assembly of the replication machinery at oriC. A central component of this assembly is the initiator protein DnaA.

No abstract

No abstract

We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the b-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The b-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the b-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We