GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2009) 14, 329-341. doi:10.1111/j.1365-2443.2008.01269.x
© 2009 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nozaki, S.
Right arrow Articles by Ogawa, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Nozaki, S.
Right arrow Articles by Ogawa, T.

Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli

 Shingo Nozaki, Yoshitaka Yamadaa and Tohru Ogawa*

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as ‘initiator titration’, a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in {Delta}datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The timing of DNA replication during the cell cycle is precisely regulated at the step of initiation. In Escherichia coli, replication starts at the chromosomal origin, oriC, once and only once, at a fixed time in the cell cycle under given steady-state growth conditions (Helmstetter 1996; Messer & Weigel 1996). The initiation timing is tightly coupled to growth rate, such that the inter-initiation time is equal to the cell doubling time. Another striking feature is that, in fast-growing cells carrying multiple copies of oriC, initiations occur synchronously from all origins within each cell.

In the initiation event, a nucleoprotein complex is formed at oriC bound by multiple molecules of the bacterial initiator protein, DnaA (Kaguni 2006; Mott & Berger 2007). As members of the complex, the DNA architectural proteins HU and integration host factor (IHF) are also important for ensuring controlled initiation events (Leonard & Grimwade 2005). In oriC, there are five 9-bp binding sites for DnaA protein (DnaA boxes R1, M, R2, R3 and R4 from the left) with a consensus sequence TTAT(C/A)CA(C/A)A (Fuller et al. 1984; Matsui et al. 1985). Several ATP–DnaA boxes have also been reported (Speck & Messer 2001; McGarry et al. 2004; Kawakami et al. 2005). Between DnaA boxes R1 and M, there is a site for the binding of IHF, which is a small, basic heterodimeric protein and among the abundant protein components organizing the bacterial nucleoid (Polaczek 1990). It is known that upon binding, IHF induces a sharp bend in DNA and regulates a variety of reactions in the recombination, transcription, replication, and partition of some plasmids (Rice et al. 1996). The IHF-binding sequence in oriC, GATCAACAACCTG, has 7 out of 9 matches to the consensus sequence recognized by IHF, WATCAANNNNTTR, where W is A or T, N is any base, and R is A or G (Friedman 1988). The binding of IHF to this site is believed to be important for the control of replication initiation because it stimulates open complex formation in vitro (Bramhill & Kornberg 1988; Hwang & Kornberg 1992), and the mutagenesis of this site in chromosomal oriC provokes moderately asynchronous and inefficient initiation in vivo (Weigel et al. 2001). Replication of minichromosomes is stimulated by IHF (Filutowicz & Roll 1990; Kano et al. 1991) and is dependent on the integrity of the IHF-binding site (Roth et al. 1994). It has been reported that IHF specifically binds to this site just at the time of initiation in the cell cycle and stimulates the redistribution of DnaA such that its binding is enhanced at weaker binding sites (R2, R3, and M as well as several non-canonical ATP–DnaA binding sites within oriC), resulting in the formation of the open complex (Cassler et al. 1995; Grimwade et al. 2000).

Following the initiation of replication, at least three different mechanisms are involved in preventing the premature reinitiation (over-initiation) of replication. In the first of these mechanisms, the nucleotide sequence GATC in E. coli DNA is methylated by DNA adenine methyltransferase (Dam) on both strands (Marinus 1996). Newly replicated GATC sites are normally remethylated within about 1 min. However, at least two of the 11 GATC sites within the 245-bp minimal oriC remain hemimethylated for approximately one-third of a generation time, due to the sequestration activities of SeqA (Campbell & Kleckner 1990; Lu et al. 1994). During this period, replication is not initiated at oriC. Second, DnaA is inactivated by a process referred to as RIDA (regulatory inactivation of DnaA) (Katayama et al. 1998). DnaA protein is active in its ATP-bound form during initiation. After initiation, the RIDA system stimulates weak ATPase intrinsic to DnaA and produces inactive ADP–DnaA due to activities of the Hda protein (Kato & Katayama 2001) and the β-sliding clamp subunit of DNA polymerase III holoenzyme.

In addition to oriC sequestration and RIDA, "initiator titration" is reported to contribute as a third mechanism to prevent over-initiation. Initiator titration is a process of the titration of DnaA molecules to newly replicated chromosomal sites containing DnaA boxes, thereby reducing the concentration of DNA-free DnaA protein below the level that triggers initiation at oriC (Hansen et al. 1991). This mechanism was originally proposed as a model, and later the datA (DnaA titration A) locus was found, which appears to function in this process (Kitagawa et al. 1998). Deletion of the datA locus resulted in extra and asynchronous initiation events, as inferred primarily from the measurement of cellular chromosome numbers after the addition of rifampicin and cephalexin, which usually inhibit new initiation and cell division, respectively (Kitagawa et al. 1998). There are about 300 DnaA boxes on the E. coli chromosome, but the datA locus appears to be solely dedicated to initiator titration, inasmuch as the simultaneous deletion of seven other strong DnaA-binding loci did not result in any alteration of replication control (Ogawa et al. 2002). The datA locus is fairly close to oriC on the E. coli genome (10 min apart on the genetic map) and should be replicated during the period of oriC sequestration. The cellular concentration of DNA-free DnaA molecules, elevated at the time of initiation, is expected to decrease by titration to the copy of newly replicated datA before the desequestration of oriC in the cell cycle. Another factor that may contribute to reducing elevated DnaA concentrations at the initiation period is the temporary inhibition of transcription of the dnaA gene just after the initiation of replication by a mechanism similar to that of oriC sequestration, which is mediated by both the GATC sequences at the promoter region as well as SeqA protein (Campbell & Kleckner 1990).

In this study, we explored the mechanism underlying DnaA titration by datA. Previous investigation has shown that two central DnaA boxes (DnaA boxes 2 and 3) in datA play a key role in initiator titration (Ogawa et al. 2002). The present results suggest that the binding of IHF to its cognate sequence located between these two DnaA boxes is pivotal in the initiator titration. The formation of a large nucleoprotein complex was suggested to be dependent on properly located DnaA boxes and the IHF-binding site in datA. Further results are presented which suggest the significance of datA in the initiation control.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Binding of IHF to datA in vitro

Within the datA locus that is approximately 1 kb in length, there are five DnaA boxes (Kitagawa et al. 1996) (Fig. 1A). Among these boxes, the second as well as the third are essential for datA function. Disruption of either of these two boxes in the chromosomal datA sequence has been suggested to provoke untimely initiation events as measured after addition of rifampicin (Ogawa et al. 2002). Therefore, it is likely that these two DnaA boxes serve as cores for the co-operative binding of large amounts of DnaA molecules to the entire datA region. However, this does not account for the observation that the region can accommodate as many as several-fold more DnaA molecules than is the oriC-mioC region, which has seven DnaA boxes within about 0.7 kb and has a lower Kd value for DnaA binding (Kitagawa et al. 1996).


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Figure 1  The datA region. (A) Genes or ORFs are indicated by grey rectangles. Bent arrows indicate transcription initiation sites. The five arrowheads indicate DnaA boxes. The datA region spans from the first to the fifth DnaA box (numbered from the left). The approximate position of DNA fragment I, used as a probe for the gel mobility-shift assay, is shown. (B) Nucleotide sequence of a part of the datA region. DnaA boxes and the IHF binding site are indicated by black boxes with arrows. Positions and sequences deleted, inserted, or displaced in this study are indicated. DnaA box 2 mutation has a 2-bp substitution in the second DnaA box and datA{Delta}box mutation has 2-bp substitutions in all five DnaA boxes.

 
Between these two DnaA boxes, there is a perfect match to the consensus sequence for binding of the protein IHF (Fig. 1B). Therefore, we investigated the possible involvement of IHF in high-capacity DnaA binding to datA. We initially assessed the ability of IHF to bind to this site in vitro using a gel mobility shift assay (Fig. 2A). Radioactively labeled fragment I, which encompasses the two essential DnaA boxes (Fig. 1A), was used as a probe. As shown in lanes 2–4, slow migrating bands appeared at the position indicated by a white arrow when the probe DNA was incubated with extracts prepared from wild-type cells. In contrast, extracts prepared from IHF-deficient cells did not cause such band shift (Fig. 2A, lanes 5–7). Furthermore, pretreatment of the wild-type extracts with an antibody preparation raised against IHF prevented such mobility shift (Fig. 2A, lanes 8–10). These results suggest that the slow migrating bands are bound by IHF. Incubation of the wild-type extracts with DNA fragments from other regions of datA did not cause any significant mobility shifting of the fragments (data not shown), suggesting that IHF binds to the cognate sequence. In addition, the results suggest that no other abundant intracellular protein specifically recognizes the datA region. At increased extract concentrations, a small amount of radioactive band shifted toward the origin, and this shift appeared to be primarily due to binding of DnaA protein, as a duplex oligonucleotide carrying the DnaA box sequence was found to inhibit the mobility shift when added as a competitor (data not shown).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Figure 2  Gel mobility-shift assay. (A) The following amounts of crude extract (Fraction I protein from wild-type or mutant cells) added were used: 0 µg (lanes 1 and 8), 0.15 µg (lanes 2 and 5), 0.6 µg (lanes 3, 6, and 9), and 2.5 µg (lanes 4, 7, and 10). (B) Assays were performed using purified IHF protein and fragment I DNA with the wild-type or core mutant sequence. The concentrations of IHF protein (nM) were 0, 5, 10, 20, 40, 60, 80, 100, and 120 for the wild-type DNA (lanes 1–9) and 0, 10, 20, 40, 80, 120, 180, 240, and 300 for the core mutant DNA (lanes 10–18). Positions of free and bound DNA are indicated with arrows.

 
The binding of IHF to its target sequence was directly examined using purified IHF protein (Fig. 2B). The gel mobility shift assay revealed that IHF binds to fragment I with an apparent Kd of approximately 8 nM. Furthermore, the introduction of a 4-bp substitution into the consensus core sequence (core mutation, Fig. 1B) remarkably reduced the binding affinity, attesting to a direct interaction with the sequence (Fig. 2B). The affinity of IHF to the datA fragment was stronger than that measured for the DNA fragment containing oriC (Kd = c. 60 nM) or attP of phage {lambda} (Kd = c. 20 nM) under similar conditions (Fig. S1 in Supporting Information). The binding of IHF was also demonstrated in a DNase I footprinting experiment (Fig. S2).

Binding of IHF to datA in vivo

To establish whether IHF is bound to datA in vivo, we modified a chromatin immunoprecipitation method (Grainger et al. 2004) by using a strain that contains the β-subunit of IHF tagged at its C-terminus with 6 x His residues. Such a strain was constructed by replacing the wild-type hip (himD) gene with the histidine-tagged version of hip on the chromosome. This strain was indistinguishable from the parental strain in terms of growth rate and cell morphology. In addition, the strain supported the replication of a pSC101-based plasmid, suggesting that activity is maintained in the tagged protein. Cells were treated with formaldehyde for the cross-linking of proteins to DNA, and the tagged protein-DNA complex was collected from the extracts using Ni-NTA agarose beads. Bound DNA was purified, and the dosage of the datA region was quantified by real-time PCR. A DNA region containing a known IHF-binding site (oriC) as well as a region lacking this site (nadD) was also examined as controls. As shown in Fig. 3, the level of IHF bound to the datA region was roughly equal to that bound to oriC. Furthermore, in cells with a core mutation in datA, binding was dramatically decreased to a background level, whereas a similar level of IHF was still bound to oriC. These data strongly suggest that IHF actually binds to datA in vivo.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Figure 3  Binding of IHF to datA and oriC in vivo. Protein-DNA complexes were cross-linked in vivo. Sonication extracts were treated with Ni-NTA agarose beads to collect the tagged IHF. Dosages of the indicated DNA loci recovered from the beads were quantified by real-time PCR. The amount of datA DNA in ihfB-6xhis cells was arbitrarily defined as 1. Averages of 4–14 assays are shown.

 
IHF binding is essential for initiation control by datA

The role played by IHF in datA function, that is, controlling replication-initiation timing, was examined by flow cytometry (Fig. 4). Exponentially growing cells were treated with rifampicin and cephalexin for several generation times to allow for the progression of replication forks to the termini while inhibiting new initiation and cell division, respectively. The number of chromosomes per cell measured is generally equal to the number of origins per cell that were present at the time of addition of the drugs.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4  Effect of various mutations in datA on initiator titration activity. Exponentially growing cells in M9 minimal medium were treated with rifampicin and cephalexin for 4–5 generations and run-out DNA histograms were obtained by flow cytometry. Mutations are described in Fig. 1B and were introduced into the chromosomal datA region.

 
As became evident from the data, increase in oriC concentration and asynchronous initiations were provoked by the core mutation. The AT cluster located upstream of the core sequence has been suggested to enhance IHF binding, but cannot support IHF binding by itself (Hales et al. 1994). The delA mutation lacking 11-bp of this region on the chromosome (Fig. 1B) did not induce the datA mutant phenotype. The DNA histogram of a strain carrying the combined mutations of the core and delA suggested that the latter mutation has a small stimulatory effect on the phenotype caused by the core mutation.

Some reports have suggested that rifampicin-resistant initiation takes place in the context of certain genetic backgrounds, including datA deletion (Hanna & Carl 1975; Hansen 1995; von Freiesleben et al. 2000; Morigen et al. 2005). Nevertheless, we employed this method, since it is a good measure of accumulated initiation potential. In this regard, we found a significant increase in oriC concentration in datA mutants in the absence of the drugs (see below).

The possibility that the binding of IHF enhances the capacity of datA for DnaA titration was examined by two methods in vivo. First, the wild-type and mutant datA sequences were tested for the derepression activity of the mioC-lacZ fusion gene, which is repressed by DnaA (Kitagawa et al. 1996) (Table 1). The β-galactosidase activity is derepressed by the wild-type datA sequence, but it is suppressed by the core mutation to a level similar to that yielded by the DnaA box 2 mutation. Therefore, it appears that the IHF binding site is required for datA to titrate DnaA. In the second method, the vector and DnaA titrating plasmids were visualized by a TetR-mCherry/tet operator system in a host strain that grows in a manner dependent on the DnaA–EYFP fusion protein in place of DnaA. As shown in Fig. 5A, most plasmids carrying a wild-type datA (pMW119-EXtetO) or core mutation (pMW119-EXcoretetO) colocalized with the fluorescence of DnaA-EYFP. The colocalized foci appeared to be located in the cytoplasm adjacent to the bulk of DnaA-EYFP which was presumably bound to the host nucleoids. On the other hand, such colocalization was much less frequent when the strain was examined that carry a plasmid bearing the DnaA box 2 mutation (pMW119-EX2tetO) or the vector plasmid (pMW119tetO). It appeared that these rare cases were due to the fact that the foci of DnaA-EYFP on the chromosome happened to colocalize with the plasmids. The intensity of the fluorescence of DnaA–EYFP on the core mutant plasmid was about one-half of that observed on the wild-type datA plasmid under conditions used for observation, whereas similar intensities of TetR-mCherry fluorescence were observed with these two plasmids (Fig. 5B). It should be noted that the measured fluorescence intensity is not proportional to the amounts of fluorescent molecules. Shorter exposure times resulted in background level of DnaA-EYFP fluorescence on pMW119-EXcoretetO, while bright foci of DnaA-EYFP were still visible on pMW119-EXtetO. These results suggest that the binding of IHF to its recognition site is necessary for high-level binding of DnaA to datA. Binding beyond a certain level of DnaA molecules is considered to be required for the proper function of datA, since weaker DnaA-EYFP foci were still detected on datA with the core mutation, where the fusion protein was presumably bound to the intact DnaA boxes.


View this table:
[in this window]
[in a new window]

  		Table 1  Derepression of MioC-β-galactosidase by DnaA protein
 

Figure 5
View larger version (66K):
[in this window]
[in a new window]

  		Figure 5  DnaA titration to datA in vivo. (A) Plasmids carrying a tetO array were visualized after a short induction of TetR-mCherry. White arrowheads indicate colocalization of DnaA–EYFP (green in colored pictures) and TetR-mCherry (magenta in colored pictures) foci. Black arrowheads with white margins indicate TetR-mCherry foci that are not colocalized with DnaA-EYFP fluorescence. Numbers of the fluorescent spots of DnaA-EYFP that colocalized with the spots of TetR-mCherry out of 100 of the latter surveyed are indicated at the left as % values. The white bar is 5 µm. A single cell or a part of a cell that is enlarged fivefold is also shown. Cells carrying the plasmid with wild-type datA (pMW119-EXtetO) were elongated due to an elevated cellular DnaA concentration (Kitagawa et al. 1998), caused by derepression of dnaA-eyfp on the chromosome. Pictures for this strain are scaled down to show enough number of cells. (B) Colocalized spots of TetR-mCherry and DnaA-EYFP were chosen at random and their fluorescence intensities were quantified. Averages of 100 (TetR-mCherry, pMW119-EXtetO), 100 (TetR-mCherry, pMW119- EXcoretetO), 335 (DnaA-EYFP, pMW119-EXtetO), and 354 (DnaA-EYFP, pMW119-EXcoretetO) foci are shown as relative values.

 
Structural requirements for datA function

Various mutations were introduced into the chromosomal datA locus to explore its essential structure for initiator titration (Fig. 1B). The mutant strains were subjected to flow cytometry analysis (Fig. 4). Deletion of a 20-bp sequence between DnaA box 2 and the IHF-binding site (delB) accompanied some asynchronous over-initiation. However, a 20-bp deletion between the IHF-binding site and DnaA box 3 (delC) had little effect on initiation control by datA. The distance between the IHF site and DnaA box 3 (84 bp) is longer than that between DnaA box 2 and the IHF site (56 bp), which may account for these observations. A combination of these two deletions (delBC) restored normal datA activity, suggesting that the relative distance between the two DnaA boxes and the IHF site in the middle is important for datA function. Strikingly, the delD mutant lacking a 30-bp sequence, including the 20-bp delC mutation, produced a DNA histogram almost identical to that of the datA deletion mutant.

Histograms typical of datA mutants were obtained when the direction of either DnaA box 2 or DnaA box 3 was reversed (inv2 or inv3, respectively). Furthermore, similar results were also obtained when a 5-bp sequence was inserted either between DnaA box 2 and the IHF site (insA) or between the IHF site and DnaA box 3 (insB). Phenotypes of these mutants were corrected to normal when an additional 5-bp sequence was inserted into the original 5-bp insertion, giving rise to a 10-bp insertion (insA+ and insB+). It should be noted that the insertion of a 5-bp or a 10-bp sequence in DNA will rotate a double helix by a half turn or a full turn, respectively.

These results suggest that, in addition to the direction of the DnaA boxes, the helical phasing and the distances between the two DnaA boxes and the IHF-binding site are important for datA to play its role in initiator titration.

Characterization of datA–DnaA interaction

To gain insight into the binding properties of DnaA to datA, we employed surface plasmon resonance (SPR) measurement. Upon increases of the ATP–DnaA concentration to 300 nM, binding to oriC approached near-saturation, whereas binding to datA did not lead to saturation at 300 nM DnaA (Fig. 6A). Unfortunately, we were unable to test DnaA concentrations above 300 nM due to an abrupt increase of response presumably caused by protein aggregation. The stoichiometry of binding at 300 nM DnaA was 28 or 25 molecules of DnaA to a single copy of datA or oriC, respectively.


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Figure 6  Analysis of the interaction between DnaA and datA/oriC by SPR (A) The amount of immobilized DNA was 48.4 RU (0.0510 fmol/mm2) for datA, and 46.3 RU (0.0957 fmol/mm2) for oriC. The concentrations of ATP–DnaA were 12.5, 25, 50, 100, 200 and 300 nM; six sensorgrams were overlaid. (B) The amount of immobilized DNA was 81.8 RU (0.0862 fmol/mm2) for datA, and 101.8 RU (0.2105 fmol/mm2) for oriC. Both forms of DnaA were injected at a concentration of 300 nM.

 
The filter-binding assay revealed a Kd value for the DnaA–datA interaction that was estimated to be twice that of the DnaA–oriC interaction (Kitagawa et al. 1996). The sensorgrams shown in Fig. 6 revealed that the lower affinity was primarily due to rapid dissociation rather than slow association.

In Fig. 6B, ADP–DnaA and ATP–DnaA were compared in terms of their affinity to datA or oriC at a protein concentration of 300 nM. Compared to ATP-DnaA, ADP–DnaA bound less efficiently to both fragments and also dissociated from them more rapidly. In addition, the reduction in affinity was less significant in the case of datA.

Cell cycle disturbance in the absence of datA function

It has been reported that over-initiation in {Delta}datA cells does not occur during exponential growth, but is induced by the addition of rifampicin (Morigen et al. 2005). This conclusion is mainly based on the kinetic analysis of DNA histograms after the addition of rifampicin. However, it remains difficult to distinguish whether extra initiations had indeed occurred after the addition of rifampicin, or if they had already occurred before the addition of rifampicin, especially in cases when they occurred less frequently than normal initiation events. In addition, it may be necessary to take into account the fact that over-initiated replication forks tend to stall near oriC (Atlung & Hansen 1993).

We employed a different approach to measure the initiation frequency. In the experiments in Table 2 and Fig. S3, a lacO array was introduced near oriC and its location was visualized by LacI-ECFP binding at three different growth phases. As summarized in Table 2, the datA{Delta}box mutant strain, carrying disruptions in all five DnaA boxes in datA, was enriched in cells with 3–4 origin foci compared to the wild type. Furthermore, decrease in cells with one origin focus and increase in cells with two origin foci were remarkable at late log to early stationary growth phases, when new initiations rarely occur. Average numbers of origin foci in the datA mutant cells were always higher than that of wild-type cells. Some foci may have contained two copies of replicated oriC which have not yet separated, especially in exponentially growing cells. However, it remains very problematic to assume that the wild-type strain is specifically enriched with such foci.


View this table:
[in this window]
[in a new window]

  		Table 2  Number of origin foci per cell
 
Consistent with the results of origin number counting, we were able to detect increased initiation frequency in datA mutants by flow cytometry (Fig. S4). The datA mutant cells had higher DNA content per cell and lower cell mass. Thus, DNA to cell mass ratio was significantly higher in the absence of datA function. The initiation of replication and cell division in these cells appear to occur at smaller cell mass stages in the cell cycle.

Taken together, these observations suggested that the absence of datA brings about increase in cellular oriC concentration and disturbs cell cycle.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The present study revealed that the tight binding of IHF to datA is essential for initiator titration. Since DNA is bent by about 160° or more upon the binding of IHF (Rice et al. 1996), DnaA molecules bound to DnaA boxes at both sides of the IHF-binding site may interact to form a large complex that is able to accommodate more DnaA molecules (Fig. 7). The datA mutant phenotype observed in some mutants with deletion, insertion or inversion (Fig. 4) might be attributed to inability to form a large titration complex due to inadequate DNA structure. The titration of a large number of DnaA molecules might be assisted by low-affinity binding sites located near canonical DnaA boxes (Fig. S2; Hansen et al. 2007). Rapid association and dissociation, as well as the more efficient titration of ADP–DnaA compared to the titration to oriC (Fig. 6), are characteristics ideally suited for the function of datA as a reservoir of DnaA molecules.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Figure 7  A model for the role played by IHF in initiator titration. A process of formation of the DnaA titration complex is shown. See text for detail.

 
It has been proposed that IHF binds to oriC only at the time of replication initiation and redistributes DnaA in oriC, leading to the formation of an open complex (Leonard & Grimwade 2005). The results of our present study suggest that IHF leads to DnaA titration to datA. Thus, IHF appears to play a key role in the localization and redistribution of DnaA to ensure the proper timing of initiation. It is tempting to speculate that IHF shuttles between datA and oriC during the E. coli cell cycle for this purpose. Further study of the dynamic behavior of proteins involved in initiation during the E. coli cell cycle will still be necessary.

Our in vitro experiments including SPR measurements did not successfully demonstrate enhanced DnaA-binding to datA by the addition of IHF (Fig. S2 and data not shown). We assume that this is the main reason for the discrepancy of the estimated titration level between the present study and the previous report based on in vivo study (Kitagawa et al. 1996). It is possible that other factors are required to reproduce the effects of IHF observed in vivo. Moreover, it may be necessary to develop means of overcoming the protein aggregation-induced limitation of the DnaA concentration to 300 nM. Factors such as DnaK, GrpE and phospholipase (Hwang et al. 1990; Hupp & Kaguni 1993) might be involved in vivo to alleviate this problem. Ordered assembly of a large titration complex might be facilitated by a factor like DiaA (Ishida et al. 2004).

The effects of increased DnaA concentrations on the initiation of DNA replication have been extensively studied employing a lacP-controlled dnaA gene (Atlung & Hansen 1993). At DnaA concentrations ranging from 1.0- to 1.5-fold of the normal level (state I), the concentration of oriC was proportional to the DnaA concentration, and the initiation synchrony was maintained. At DnaA levels ranging from 1.5- to 3-fold above the normal level (state II), the concentration of oriC increased significantly, but the rate of fork movement was severely reduced, especially near the origin. Further increases in the DnaA concentration (state III) resulted in a decreased oriC concentration, as measured by Southern hybridization, but the number of oriC per cell estimated by flow cytometry of rifampicin-treated samples remained high, suggesting rifampicin-resistant initiation. Judging from the flow cytometry data and the observation that the DnaA concentration in {Delta}datA cells was not measurably greater than that in wild-type cells (unpublished data), the concentration of cellular DnaA during the cell cycle of {Delta}datA cells appeared to oscillate around state I, close to state II, or at the boundary between these two states. Thus, the disturbance of DNA replication physiology in these cells was not as marked as that previously seen in hda or seqA cells (Lu et al. 1994; Kato & Katayama 2001). We assume that DnaA titration to datA is essential in the fine-tuning of initiation timing during some period before initiation, that is, when ATP–DnaA level is high but oriC sequestration and RIDA are inoperative. In other period during the cell cycle, DnaA titration to datA may serve to complement other systems or as a fail-safe system.

It is likely that the initiator titration mechanism is solely dependent on the datA locus, as disruption of high-affinity sites for DnaA on the E. coli chromosome, with the exception of datA, had no effect on initiation control, even when seven such sites were disrupted simultaneously (Ogawa et al. 2002). At present, it remains unclear how such an initiator titration system is conserved among bacterial species. datA is located in the glyVXY–yjeS region (95 min) on the E. coli genetic map. The DNA sequence of this region is not well-conserved, even among bacterial species that belong to the gamma subclass of proteobacteria, except for those closely related to E. coli such as Shigella flexneri and Salmonella typhymurium. It may be possible that, in other bacteria, a unique DnaA box cluster plays a role in initiator titration in a sequence that differs substantially from E. coli datA.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Bacterial strains and plasmids

The E. coli strains used to prepare crude extracts for the gel mobility shift assay were K91 (Hfr phoA6 tonA22 garB10 ompF627 relA1 pit-10 spoT1 T2R P02A) and K1174 [K91 himA [DE(82::Tn10)] hip[DE(3::CamR)]]. These strains were obtained from the E. coli Strain National BioResource Project of the National Institute of Genetics, Mishima, Japan. Escherichia coli strains carrying mutations in datA were derivatives of W3110 [IN(rrnD-rrnE)1 rph-1]. Mutations were created in vitro by the overlap extension PCR method (Sambrook & Russell 2001) using the DNA of Kohara's {lambda}-clone 652 (Kohara et al. 1987) as a template. The mutagenic primers and the forward and reverse primers (SSPR and BSPL, respectively) used to amplify the datA region are listed in Table S1 in Supporting Information. The high-fidelity enzyme, KOD-Plus-DNA polymerase (TOYOBO) was used in the PCR reactions. The PvuII-XhoI fragments (1872 bp) encompassing datA were obtained from the PCR products and blunt-end ligated into the BamHI site of pKH5002SB (Kitagawa et al. 1998). Displacement of the chromosomal wild-type datA sequence by the mutant sequence was carried out as previously described (Kitagawa et al. 1998), and mutants were selected by restriction enzyme analyses of the PCR products. The datA mutations core+delA, delBC, insA+, and insB+ were constructed on pKH5002SB carrying the PvuII-XhoI fragments with mutations of core, delB, insA, and insB, respectively, and the mutations were transferred to the chromosome of W3110, as described above. RSD448 (= W3110{Delta}datA:: kan) (Kitagawa et al. 1998) and MC1061 (Kitagawa et al. 1996) have previously been described. ON373 (= MG1655hip-6his), a strain expressing IHFβ protein tagged with 6xHis at the C-terminus via four amino acid residues (SGSG), was constructed as previously described (Uzzau et al. 2001). A plasmid template, pSUB7, and the primers HIPHTAG and P2HIP (Table S1) were used for PCR, and the transformant was constructed as previously described (Datsenko & Wanner 2000). MG1655core was constructed as described for W3110core, and MG1655core hip-6his (= ON374) was constructed by P1 transduction. The datA{Delta}box1–5 mutation (Ogawa et al. 2002) carrying base substitutions in all five DnaA boxes in datA is referred to as datA{Delta}box in this article. The strains used for the experiments in Fig. 5 were MG1655 dnaA-eyfp {Delta}(353820–353822)::(araC pBAD-tetR-mCherry) carrying pMW119tetO array, pMW119-EXtetO array, pMW119-EX2tetO array or pMW119-EXcoretetO array. In this host strain, a portion of the chromosomal dnaA gene encoding the 87–104 residues of DnaA was replaced by the eyfp-encoding sequence. The strains used for the experiments in Table 2 were MG1655 {Delta}(353820–353822):: (araC pBAD-lacI-ecfp) attTn7:: lacO array, which was derived from TH130 (Hatano et al. 2007) and MG1655 {Delta}(353820–353822):: (araC pBAD-lacI-ecfp) attTn7:: lacO array datA{Delta}box. The construction of the strains used for fluorescence microscopy will be described in detail elsewhere. Plasmids pMW119, pMW119-EX, and pMW119-EX2 are derivatives of pSC101 and have been described (Kitagawa et al. 1998). pMW119-EXcore carries the core mutation (Fig. 1B) in pMW119-EX. pAML1 is a pACYC184-based plasmid carrying the mioC-lacZ fusion gene (Kitagawa et al. 1998). Plasmids pSUB7 (Uzzau et al. 2001), pKD46, and pCP20 (Datsenko & Wanner 2000) have also been described previously.

Gel mobility-shift assay

The probe DNA (Fragment I) was prepared by PCR using {lambda} clone 652 DNA or pKH5002SB DNA carrying the datA PvuII-XhoI fragments with the core mutation as templates, and ASEI and ORF51 (Table S1) as primers. The PCR products were digested with BsaHI and subjected to agarose gel electrophoresis. The isolated 289-bp fragments were labeled using [{alpha}-32P]dCTP and the large fragment of DNA polymerase I to achieve a specific activity of 580 cpm/fmol. For the assay in Fig. 2A, crude extracts (Fraction I) were prepared from harvested cells grown to a mid-log phase by lysozyme treatment followed by freezing in liquid nitrogen, thawing at room temperature, and high-speed centrifugation. The reaction mixtures (10 µL) contained 20 mM Tris–HCl (pH 7.5), 75 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, 10% (v/v) glycerol, 100 µg/mL poly(dI)·poly(dC), 1.7 nM probe fragments, and the indicated amounts of Fraction I or purified IHF protein. In cases when the antibody against IHF was added, it was pre-incubated with Fraction I for 5 min at 30 °C before the addition of other components. After being incubated at 30 °C for 15 min, the reaction mixtures were chilled on ice and subjected to 5% polyacrylamide gel electrophoresis at 4 °C in a solution of 50 mM Tris–borate (pH 8.3) and 1 mM EDTA. After electrophoresis, the gel was dried on Whatman DE81 paper, and radioactive bands were detected using a BAS 1000 Bio-Imaging Analyzer (Fuji Photo Film).

Isolation of DNA fragments bound to IHF in vivo and real-time PCR quantification

Cross-linking of proteins to DNA and the preparation of cell extracts were performed essentially as previously described (Grainger et al. 2004), except that sonication was carried out in 50 mM Na-PO4, 10 mM imidazole, 5 mM Tris–HCl, 0.3 M NaCl, 1 mg/mL lysozyme, and 1x complete mini without EDTA (Roche Applied Science), pH 8.0. A 250-µL cell extract (approximately 5 x 108 cells equivalent) was mixed with a 50-µL slurry (50%) of Ni-NTA agarose (Qiagen) in the same buffer (lacking lysozyme and complete mini), and the samples were mixed gently for 60 min at 4 °C. Materials bound to the agarose beads were recovered as recommended by the manufacturer, and the buffer was exchanged for a solution of 10 mM Tris–HCl, 1 mM EDTA, and 0.1% SDS (pH 7.5) by passage through a Microspin S-400 column (GE Healthcare Bioscience). Samples were then treated with 0.4 mg/ml proteinase K for 2 h at 42 °C and then for 12 h at 65 °C for uncrosslinking. DNA was purified by phenol-chloroform treatment and ethanol precipitation in the presence of 5 µg of glycogen. Real-time PCR was performed using iQ SYBR Green Supermix and an iCycler iQ (Bio-Rad Laboratories). The primers used were RTYBENR and RTYBENL for the nadD locus, RTDATAR and RTDATAL for the datA locus, and RTORICR and RTORICL for the oriC locus (Table S1). The datA region amplified by PCR was a 103-bp fragment encompassing DnaA box 2 and was located 15 bp away from the IHF recognition sequence. Four tubes containing 10-fold serially diluted DNA of E. coli MG1655 (0.01 to 10 ng) were included as standards in each PCR run. Starting quantity of each sample was measured relative to this standard DNA.

Flow cytometry

Cells were grown in M9 minimal medium containing 0.2% glucose (Miller 1972), except where indicated. The number of oriC per cell was determined by flow cytometry as the number of chromosomes per cell after the completion of ongoing replication (Løbner-Olesen et al. 1989; Kitagawa et al. 1998). Cells fixed in 70% ethanol (0.002 OD600 units) were washed once with 10 mM Tris–HCl (pH 7.5) and 1 mM EDTA, and were then suspended in 20 µL of 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 50 µg/mL RNase I. After incubation for 30 min at 37 °C, the suspension was mixed with 1 mL of a solution that consisted of 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.05% (v/v) Triton X-100, and 0.33 µL of a 10-fold diluted solution of PicoGreen (Molecular Probes). Flow cytometry was carried out with FACSCalibur (Beckton Dickinson).

Microscopy

A portion of a culture was withdrawn and concentrated by brief centrifugation and suspension in fresh medium at room temperature. A 10-µL aliquot was mounted on a poly-L-lysine-coated slide glass and subjected to microscopic observations using an Axiovert 200 M inverted microscope (Carl Zeiss) equipped with appropriate filter sets and MetaMorph ver. 6.1 software (MDS). For the observation of two fluorescent colors, cells of MG1655dna A-eyfp {Delta}(353820–353822)::(araC pBAD- tetR-mcherry) carrying the pMW119 tetO-based plasmids were grown overnight at either at 30 °C or at 37 °C in M9 glucose medium containing 30 µg/mL ampicillin and 10 µg/mL gentamicin, and the culture was inoculated into fresh medium without drugs at OD650 = 0.03. After 3–4 h of cultivation, the cells were collected by centrifugation at room temperature, resuspended in the same volume of M9 medium containing 0.2% L-arabinose in the place of glucose, and cultivated for an additional 90 min to induce TetR-mCherry. This short-duration induction was necessary to avoid growth inhibition. Cultivation temperature (30 °C or 37 °C) did not affect the results. Exposure times for observation of fluorescence were fixed (4 s for EYFP and 5 s for mCherry) in all experiments and the quantification of fluorescence intensity was performed using MetaMorph software.

Measurement of SPR

To prepare DNA fragments for the interaction assay, the HindIII-SacI fragment of M13-EX carrying datA and the AatII-ClaI fragment spanning oriC were labeled at the 3' end with biotin-16-ddUTP by terminal deoxynucleotidyltransferase, followed by treatment with SalI or XhoI, respectively, and isolation of the larger DNA fragments. DNA was purified and immobilized to the streptoavidin-coupled Pioneer Sensor Chip F1 (Biacore) according to the manufacturer's instructions. DnaA protein was purified as described previously (Kitagawa et al. 1996), with the exception that the final step of gel filtration was performed using ProteinPak G-300 (Waters) and a buffer consisting of 50 mM HEPES–KOH (pH 7.6), 10 mM Mg(OAc)2, 2 mM dithiothreitol, 0.1 mM EDTA, 300 mM potassium glutamate, and 15% glycerol. Solutions of ATP–DnaA and ADP–DnaA included the respective nucleotides at a concentration of 100 µM. Measurements were performed with Biacore 2000 (Biacore) at 25 °C and at a flow rate of 25 µL/min in a buffer consisting of 20 mM HEPES–KOH (pH 8.0), 5 mM Mg(OAc)2, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 100 mM potassium glutamate, 100 µM ATP (or ADP in case of measurements with ADP–DnaA), 10 µg/mL bovine serum albumin, and 0.005% Tween 20. DnaA protein was injected at time zero in the same buffer containing 1 µg/mL each of poly (dA-dT) and poly (dI-dC). The injection period and the dissociation period each lasted 4 min. At the end of each cycle, the sensor chips were washed with 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.3 M NaCl, and 0.05% SDS for 1 min. Correction for solvent effects was performed as described elsewhere (Frostell-Karlsson et al. 2000).

Other materials and methods

Assay for β-galactosidase was carried out as described (Kitagawa et al. 1998). Restriction enzymes and enzymes used for molecular cloning were from New England Biolabs or Takara Biochemicals, except where indicated. DNase I was from Takara Biochemicals. DnaA protein was prepared as described (Kitagawa et al. 1996). Purified IHF protein and antibody against IHF were gifts from AB Oppenheim (The Hebrew University of Jerusalem, Israel) and A Ishihama (Hosei University, Koganei, Japan), respectively. Standard methods were used as described for DNA manipulations (Sambrook & Russell 2001).


   Acknowledgements
 
Authors thank A Oppenheim for IHF protein, A Ishihama for antibody against IHF protein, S Uzzau, BL Wanner, T Hatano and H Niki (National Institute of Genetics, Mishima, Japan) for strains and plasmids, and National BioResource Project at National Institute of Genetics for strains. This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science.


   Footnotes
 
Communicated by: Hiroyuki Araki

aPresent address: Coviden Japan, 1217-1 Tomonaga, Fukuroi, Shizuoka 437-0004, Japan. Back

* Correspondence: h44851a{at}cc.nagoya-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Atlung, T. & Hansen, F.G. (1993) Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli. J. Bacteriol. 175, 6537–6545.[Abstract/Free Full Text]

Bramhill, D. & Kornberg, A. (1988) Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52, 743–755.[CrossRef][Medline]

Campbell, J.L. & Kleckner, N. (1990) E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell 62, 967–969.[CrossRef][Medline]

Cassler, M.R., Grimwade, J.E. & Leonard, A.C. (1995) Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin. EMBO J. 14, 5833–5841.[Medline]

Datsenko, K.A. & Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645.[Abstract/Free Full Text]

Filutowicz, M. & Roll, J. (1990) The requirement of IHF protein for extrachromosomal replication of the Escherichia coli oriC in a mutant deficient in DNA polymerase I activity. New Biol. 2, 818–827.[Medline]

Friedman, D.I. (1988) Integration host factor: A protein for all reasons. Cell 55, 545–554.[CrossRef][Medline]

Frostell-Karlsson, Å., Remaeus, A., Roos, H., Andersson, K., Borg, P., Hämäläinen, M. & Karlsson, R. (2000) Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels. J. Med. Chem. 43, 1986–1992.[CrossRef][Medline]

Fuller, R.S., Funnell, B.E. & Kornberg, A. (1984) The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38, 889–900.[CrossRef][Medline]

Grainger, D.C., Overton, T.W., Reppas, N., Wade, J.T., Tamai,, E., Hobman, J.L., Constantinidou, C., Struhl, K., Church, G. & Busby, S.J.W. (2004) Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays. J. Bacteriol. 186, 6938–6943.[Abstract/Free Full Text]

Grimwade, J.E., Ryan, V.T. & Leonard, A.C. (2000) IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli. Mol. Microbiol. 35, 835–844.[CrossRef][Medline]

Hales, L.M., Gumport, R.I. & Gardner, J.F. (1994) Determining the DNA sequence elements required for binding integration host factor to two different target site. J. Bacteriol. 176, 2999–3006.[Abstract/Free Full Text]

Hanna, M.H. & Carl, P.L. (1975) Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of E. coli: role of ribonucleic acid synthesis, protein synthesis, and cell division. J. Bacteriol. 121, 219–226.[Abstract/Free Full Text]

Hansen, F.G. (1995) Reinitiation kinetics in eight dnaA (Ts) mutants of Escherichia coli: rifampicin resistant initiation of chromosome replication. Mol. Microbiol. 15, 133–140.[CrossRef][Medline]

Hansen, F.G., Christensen, B.B. & Atlung, T. (1991) The initiator titration model: computer simulation of chromosome and minichromosome control. Res. Microbiol. 142, 161–167.[Medline]

Hansen, F.G., Christensen, B.B. & Atlung, T. (2007) Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication initiator protein DnaA in E. coli. J. Mol. Biol. 367, 942–952.[CrossRef][Medline]

Hatano, T., Yamaichi, Y. & Niki, H. (2007) Oscillating focus of SopA associated with filamentous structure guides partitioning of F plasmid. Mol. Microbiol. 64, 1198–1213.[CrossRef][Medline]

Helmstetter, C. (1996) Timing of synthetic activities in the cell cycle. In: Escherichia coli and Salmonella (eds F.C. Neidhardt, R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter & H.E. Umbarger), pp. 1627–1639. Washington DC: ASM Press.

Hupp, T.R. & Kaguni, J.M. (1993) Activation of DnaA5 protein by GrpE and DnaK heat shock proteins in initiation of DNA replication in Escherichia coli. J. Biol. Chem. 268, 13137–13142.[Abstract/Free Full Text]

Hwang, D.S., Crooke, E. & Kornberg, A. (1990) Aggregated DnaA protein is dissociated and activated for DNA replication by phospholipase or DnaK protein. J. Biol. Chem. 265, 19244–19248.[Abstract/Free Full Text]

Hwang, D.S. & Kornberg, A. (1992) Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF. J. Biol. Chem. 267, 23083–23086.[Abstract/Free Full Text]

Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M., Kubota, T., Ogata, Y., Sekimizu, K. & Katayama, T. (2004) DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication. J. Biol. Chem. 279, 45546–45555.[Abstract/Free Full Text]

Kaguni, J.M. (2006) DnaA: controlling the initiation of bacterial DNA replication and more. Annu. Rev. Microbiol. 60, 351–375.[CrossRef][Medline]

Kano, Y., Ogawa, T., Ogura, T., Hiraga, S., Okazaki, T. & Imamoto, F. (1991) Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli. Gene 103, 25–30.[CrossRef][Medline]

Katayama, T., Kubota, T., Kurokawa, K., Crooke, E. & Sekimizu, K. (1998) The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase. Cell 94, 61–71.[CrossRef][Medline]

Kato, J. & Katayama, T. (2001) Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli. EMBO J. 20, 4253–4262.[CrossRef][Medline]

Kawakami, H., Keyamura, K. & Katayama, T. (2005) Formation of an ATP–DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family. J. Biol. Chem. 280, 27420–27430.[Abstract/Free Full Text]

Kitagawa, R., Mitsuki, H., Okazaki, T. & Ogawa, T. (1996) A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome. Mol. Microbiol. 19, 1137–1147.[CrossRef][Medline]

Kitagawa, R., Ozaki, T., Moriya, S. & Ogawa, T. (1998) Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein. Genes Dev. 12, 3032–3043.[Abstract/Free Full Text]

Kohara, Y., Akiyama, K. & Isono, K. (1987) The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50, 495–508.[CrossRef][Medline]

Leonard, A.C. & Grimwade, J.E. (2005) Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding. Mol. Microbiol. 55, 978–985.[CrossRef][Medline]

Løbner-Olesen, A., Skarstad, K., Hansen, F.G., von Meyenburg, K. & Boye, E. (1989) The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell 57, 881–889.[CrossRef][Medline]

Lu, M., Campbell, J.L., Boye, E. & Kleckner, N. (1994) SeqA: a negative modulator of replication initiation in E. coli. Cell 77, 413–426.[CrossRef][Medline]

Marinus, M.G. (1996) Methylation of DNA. In: Escherichia coli and Salmonella (eds F.C. Neidhardt, R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter & H.E. Umbarger), pp. 782–791. Washington DC: ASM Press.

Matsui, M., Oka, A., Takanami, M., Yasuda, S. & Hirota, Y. (1985) Sites of dnaA protein-binding in the replication origin of the E. coli K-12 chromosome. J. Mol. Biol. 184, 529–533.[CrossRef][Medline]

McGarry, K.C., Ryan, V.T., Grimwade. J.E. & Leonard, A.C. (2004) Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA–ATP. Proc. Natl Acad. Sci. USA 101, 2811–2816.[Abstract/Free Full Text]

Messer, W. & Weigel, C. (1996) Initiation of chromosome replication. In: Escherichia coli and Salmonella (eds F.C. Neidhardt, R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter & H.E. Umbarger), pp. 1579–1601. Washington DC: ASM Press.

Miller, J.H. (1972) Experiments in Molecular Genetics. New York: Cold Spring Harbor Laboratory Press.

Morigen, Molina, F. & Skarstad, K. (2005) Deletion of the datA site does not affect once-per-cell-cycle timing but induces rifampin-resistant replication. J. Bacteriol. 187, 3913–3920.[Abstract/Free Full Text]

Mott, M.L. & Berger, J.M. (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol. 5, 343–354.[CrossRef][Medline]

Ogawa, T., Yamada, Y., Kuroda, T., Kishi, T. & Moriya, S. (2002) The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli. Mol. Microbiol. 44, 1367–1375.[CrossRef][Medline]

Polaczek, P. (1990) Bending of the origin of replication of E. coli by binding of IHF at a specific site. New Biol. 2, 265–271.[Medline]

Rice, P.A., Yang, S., Mizuuchi, K. & Nash, H.A. (1996) Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 87, 1295–1306.[CrossRef][Medline]

Roth, A., Urmoneit, B. & Messer, W. (1994) Functions of histone-like proteins in the initiation of DNA replication at oriC of Escherichia coli. Biochimie 76, 917–923.[CrossRef][Medline]

Sambrook, J. & Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. New York: Cold Spring Harbor Laboratory Press.

Speck, C. & Messer, W. (2001) Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA. EMBO J. 20, 1469–1476.[CrossRef][Medline]

Uzzau, S., Figueroa-Bossi, N., Rubino, S. & Bossi, L. (2001) Epitope tagging of chromosomal genes in Salmonella. Proc. Natl Acad. Sci. USA 98, 15264–15269.[Abstract/Free Full Text]

von Freiesleben, U., Rasmussen, K.V., Atlung, T. & Hansen, F. (2000) Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants. Mol. Microbiol. 37, 1087–1093.[CrossRef][Medline]

Weigel, C., Messer, W., Preiss, S., Welzeck, M. & Boye, E. (2001) The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome. Mol. Microbiol. 40, 498–507.[CrossRef][Medline]

Received: 21 October 2008
Accepted: 24 November 2008




This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
S. Nozaki, H. Niki, and T. Ogawa
Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle
J. Bacteriol., August 1, 2009; 191(15): 4807 - 4814.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nozaki, S.
Right arrow Articles by Ogawa, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Nozaki, S.
Right arrow Articles by Ogawa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS