Erik Boye

SeqA: A negative modulator of replication initiation in E. coli

In E. coli, replication initiates at a genetically unique origin, oriC. Rapidly growing cells contain multiple oriC copies. Initiation occurs synchronously, once and only once per cell cycle at all origins present. Secondary initiations are prevented by a sequestration process that acts uniquely on newly replicated origins, which are marked because they are hemimethylated at GATC sites. We report the identification of a gene required for sequestration and demonstrate that this gene, seqA, also serves as a negative modulator of the primary initiation process. All previously identified in vivo initiation factors play positive roles. Thus, precise control of replication initiation may involve a balance between positive and negative elements. We suggest that SeqA might be a cooperativity factor, acting to make the replication initiation process dependent upon cooperative interactions among components.

Timing of initiation of chromosome replication in individual Escherichia coli cells

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.

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

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.

The DnaA protein determines the initiation mass of Escherichia coli K-12

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

The role of dam methyltransferase in the control of DNA replication in E. coli

The timing and control of initiation of DNA replication in E. coli was studied under conditions where the cellular level of dam methyltransferase was controlled by a temperature-inducible promoter. Flow cytometry was used to demonstrate that the synchrony of initiation at the several origins within each cell was critically dependent on the level of dam methyltransferase. Initiations were shown to be synchronous only in a narrow temperature range. The data are explained by a model where a newly replicated and therefore hemimethylated oriC is inert for reinitiation. Such a model may be applicable to eukaryotic cells, where classes of origins are initiated in synchrony and only once per cell cycle.

Limiting DNA replication to once and only once.

In Escherichia coli cells, the origin of chromosomal replication is temporarily inactivated after initiation has occurred. Origin sequestration is the first line of defence against over‐initiation, providing a time window during which the initiation potential can be reduced by: (i) titration of DnaA proteins to newly replicated chromosomal elements; (ii) regulation of the activity of the DnaA initiator protein; and (iii) sequestration of the dnaA gene promoter. This review represents the first attempt to consider together older and more recent data on such inactivation mechanisms in order to analyze their contributions to the overall tight replication control observed in vivo. All cells have developed mechanisms for origin inactivation, but those of other bacteria and eukaryotic cells are clearly distinct from those of E. coli. Possible differences and similarities are discussed.

The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate

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 have used flow cytometry to measure the initiation mass of E. coli K‐12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonically with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication.

Flow cytometry of bacteria: A promising tool in experimental and clinical microbiology

The DNA and protein content of individual Escherichia coli cells were measured at a rate of 10(4) cells per second with a sensitive microscope-based flow cytometer. DNA and protein were quantified by measuring the fluorescence from cells stained with a combination of the DNA-binding drugs Mithramycin and ethidium bromide and by scattered light, respectively. Separate experiments demonstrated that the light scatter signal was proportional to protein content. Dual parameter histograms (fluorescence/scattered light) of bacterial cultures gave detailed pictures of changes dependent upon the growth conditions and of the cell cycle kinetics. Effects of antibiotics could be readily detected and characterized after a few hours. The results demonstrate that flow cytometry is a promising method for application in experimental and clinical microbiology.

DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1

Cdt1 is an essential protein required for licensing of replication origins. Here, we show that in Schizosaccharomyces pombe, Cdt1 is proteolysed in M and G1 phases in response to DNA damage and that this mechanism seems to be conserved from yeast to Metazoa. This degradation does not require Rad3 and Cds1, indicating that it is independent of classic DNA damage and replication checkpoint pathways. Damage‐induced degradation of Cdt1 is dependent on Cdt2 and Ddb1, which are components of a Cul4 ubiquitin ligase. We also show that Cdt2 and Ddb1 are needed for cell‐cycle changes in Cdt1 levels in the absence of DNA damage. Cdt2 and Ddb1 have been shown to be involved in the degradation of the Spd1 inhibitor of ribonucleotide reductase after DNA damage, and we speculate that Cdt1 downregulation might contribute to genome stability by reducing demand on dNTP pools during DNA repair.

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

In wild‐type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two‐ to threefold relative to wild type. Here, we show that the DnaA protein concentration was two‐ to threefold lower in the dnaA204 mutant compared with the wild‐type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild‐type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half‐life of the protein. Our results suggest that the DnaA204 protein has essentially wild‐type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA–DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.