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Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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As the two replication forks generated from one origin replicate bidirectionally along the chromosome, there is a transient wave of hemimethylation behind the forks because the newly replicated daughter strands are unmethylated at GATC sites for a period of time before Dam methyltransferase methylates them (at the N6 position of adenine). SeqA protein interacts with hemimethylated DNA, and prevents newly replicated origins from being initiated (Slater et al. 1995).
SeqA consists of two functionally distinct domains (Guarnéet al. 2002; Fujikawa et al. 2003). The C-terminal domain is responsible for interaction with DNA and the N-terminus has the ability to form multimers (Guarnéet al. 2002; Fujikawa et al. 2003). SeqA requires two hemimethylated GATC sites located on approximately the same face of the DNA helix for stable binding (Brendler & Austin 1999; Brendler et al. 2000), and probably binds this pair of sites as a dimer (Guarnéet al. 2002; Kang et al. 2005). SeqA also binds specifically, with lower affinity, to fully methylated origin DNA (Slater et al. 1995; Skarstad et al. 2000). For this binding, several GATC sites or, alternatively, other determinants in the left part of oriC, seem to be required (Slater et al. 1995; Skarstad et al. 2000). For full sequestration of hemimethylated DNA an oriC-like distribution of GATC sites is sufficient (Bach & Skarstad 2005).
SeqA has been found to localize as discrete foci in the cell, also in the absence of oriC (Hiraga et al. 1998; Onogi et al. 1999). The formation of SeqA foci is dependent upon Dam methylation and ongoing DNA replication and the pattern of migration of SeqA foci is different from the pattern of oriC migration (Hiraga et al. 1998; Onogi et al. 1999; Bates & Kleckner 2005). This suggests that the foci represent multimers of SeqA bound to the hemimethylated DNA right behind the replication forks (Brendler et al. 2000; Hiraga et al. 2000). This higher-order structure formed by SeqA may be important for the pairing up of replisomes into replication-factories and possibly for the formation of so-called hyperstructures (Norris et al. 2002; Molina & Skarstad 2004). The lack of SeqA leads to overinitiation, to increased negative supercoiling of chromosomal and plasmid DNA (Weitao et al. 1999, 2000), and to aberrant segregation of nucleoids and filamentation (Lu et al. 1994; von Freiesleben et al. 2000). Over-expression of SeqA delays nucleoid segregation and cell division (Bach et al. 2003). In vitro, SeqA has been found to generate positive supercoils in plasmid DNA (Klungsøyr & Skarstad 2004) and to build a higher-order structure that restrains negative supercoils (Torheim & Skarstad 1999; Klungsøyr & Skarstad 2004). These findings indicate that SeqA may contribute to the organization of newly replicated DNA at the replication forks, and possibly to the process of sister chromosome segregation (Onogi et al. 1999; Brendler et al. 2000; Hiraga et al. 2000).
In a genetic screen for sequestration-deficient mutants, several seqA mutants were isolated (Lu et al. 1994; von Freiesleben et al. 1994). One of these, the seqA4 mutant, has a point mutation that changes amino acid 25 from alanine to threonine in the N-terminal multimerization domain (Fossum et al. 2003). Here, we have characterized the mutant SeqA4 protein to gain more insight into how the SeqA protein multimerizes into higher-order structures, and whether the multimeric state of the protein is required for origin sequestration and organization of newly replicated DNA.
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Results |
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The SeqA protein can be proteolytically cleaved into C- and N-terminal domains. Gel filtration analysis has revealed that the C-terminal domain (amino acid residues 51–181) elutes as a monomer, whereas the N-terminal domain (amino acid residues 1–50) elutes as an aggregate (Guarnéet al. 2002; Fujikawa et al. 2003). We investigated whether the point mutation in the SeqA4 N-terminus affects its ability to multimerize. The SeqA and SeqA4 proteins were cleaved by trypsin, and the resulting C- and N-terminal domains were separated by gel filtration on a Superose 12 HR 10/30 column (Fossum et al. 2003). In accordance with previous reports (Guarnéet al. 2002; Fujikawa et al. 2003), the C-terminal domain eluted as a monomer (data not shown) and the wild-type N-terminal domain eluted as an aggregate of around octamer size (40–50 kDa; Fig. 1A). The SeqA4 N-terminal domain did not multimerize. It eluted at slightly above monomer size (5–10 kDa; Fig. 1A), and thus was in monomer or dimer form. The result shows that the SeqA4 N-terminus is deficient in multimerization.
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SeqA4 forms a basic binding unit capable of binding two hemimethylated GATC sites with wild-type affinity
We then investigated whether the SeqA4 protein's deficiency in multimerization affected its ability to bind to DNA. An end-labeled, hemimethylated oriC fragment with 11 GATC sites, oriC20 (Fossum et al. 2003), was incubated with SeqA4 or SeqA protein (1–250 nM) in the presence of competitor DNA and analyzed by gel electrophoresis. At increasing concentrations of SeqA4 protein, an increasing ladder of band shifts was seen (complexes I–IV; Fig. 2A, lanes 9–15). In contrast, only two bands (complexes I and II, Fig. 2A) were apparent after incubation of oriC20 with wild-type SeqA protein; the rest of the shifted species were seen as large complexes trapped in the wells (complex M, Fig. 2A), in accordance with previous results (Fossum et al. 2003; Han et al. 2003, 2004). Complexes I and II presumably represent one and two SeqA dimers, respectively, bound to one and two pairs of GATC sites. Complexes III and IV probably represent three and four SeqA4 dimers bound to three and four pairs of GATC sites. The DNA fragment has 11 GATC sites. The reason for not finding a complex V with five SeqA4 dimers bound to five pairs of GATC sites is not known, but could have to do with nonoptimal spacing of some of the GATC sites. The finding that SeqA4 formed complexes III and IV and not complex M, can be explained by the inability of SeqA4 protein to multimerize into large complexes via N-terminal interactions (Fig. 1). Quantification of the amount of free fragment revealed that SeqA and SeqA4 proteins bound with similar affinities to hemimethylated oriC20 fragments (Fig. 2B). Thus, the SeqA4 protein did not form higher-order structures, but the specific binding of SeqA4 dimers to pairs of hemimethylated sites was similar to that of the wild-type protein, leading to 50% shift at about 10 nM protein.
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Binding of SeqA4 protein to DNA generates positive supercoils
Wild-type SeqA protein has the ability to affect plasmid topology (Torheim & Skarstad 1999; Klungsøyr & Skarstad 2004). When incubated with nicked plasmid, it binds the DNA in such a way that positive supercoils are trapped after religation (Klungsøyr & Skarstad 2004). Here, we investigated whether mutant SeqA4 protein would produce the same effect. Fully methylated nicked pOC170 plasmid (4 nM) was incubated with SeqA or SeqA4 (0.45–2.4 µM) and subsequently treated with DNA ligase. The resulting topoisomers were separated in 1% agarose (Fig. 3A) (Klungsøyr & Skarstad 2004). In the presence of either SeqA or SeqA4 (Fig. 3A, lanes 3–7), supercoils were trapped by ligation. At 120–300 SeqA dimers per plasmid, up to eight supercoils were introduced (Fig. 3A, left panel, lanes 4–7) in accordance with earlier results (Klungsøyr & Skarstad 2004). At 55–170 SeqA4 dimers per plasmid, up to ten supercoils were introduced (Fig. 3A, right panel, lanes 3–5). The supercoils generated by the binding of wild-type SeqA have been shown to be positive (Klungsøyr & Skarstad 2004). Gel electrophoresis in agarose containing a low amount of chloroquine (1 µg/mL; see Experimental procedures) revealed that the supercoils generated by the binding of SeqA4 are also positive (data not shown). The results show that a SeqA4 dimer binds to the DNA in a fashion similar to that of a wild-type SeqA dimer, and also that the generation of positive supercoils by SeqA does not require formation of higher-order structures as shown schematically in Fig. 3B.
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The SeqA protein has been found to affect the topology of negatively supercoiled plasmids in two different ways (Klungsøyr & Skarstad 2004). First, as described previously, the binding causes a topology change equivalent to that of a limited number of positive supercoils. Second, at higher concentrations, SeqA forms a structure that seems to affect the DNA in the "opposite direction" and leads to restraint of most of the negative supercoils. The latter effect was detected by the failure of calf thymus topoisomerase I (Topo I) to relax the negative supercoils (Torheim & Skarstad 1999; Klungsøyr & Skarstad 2004). It was suggested that the formation of a higher-order structure led to restraint of negative supercoils, or alternatively, that the structure impaired the access of Topo I to DNA (Klungsøyr & Skarstad 2004).
Here, fully methylated, negatively supercoiled pBSoriC plasmid (4 nM) was incubated with SeqA or SeqA4 (0.45–2.8 µM) and subsequently treated with Topo I (Klungsøyr & Skarstad 2004). The resulting topoisomers were separated on agarose gels containing no or a high level (40 µg/mL) of chloroquine. At high concentrations of SeqA (around 300 SeqA dimers per plasmid), plasmids remained relatively unchanged after treatment with Topo I (supercoils were restrained) (Fig. 4A, compare lanes 1 and 8), in accordance with earlier results (Torheim & Skarstad 1999; Klungsøyr & Skarstad 2004). In contrast, in the presence of mutant SeqA4 protein, all negative supercoils were relaxed and up to six positive supercoils introduced (Fig. 4B). The positive nature of the supercoils was determined by gel electrophoresis in agarose containing a low amount of chloroquine (1 µg/mL; data not shown). This result shows that binding of SeqA4 protein does not impair the relaxation of negative supercoils performed by Topo I (shown schematically in Fig. 4D). Because SeqA4 does not form large multimers, the result supports the notion that multimerization by wild-type SeqA leads to restraint of negative supercoils (Fig. 4C). The result also shows that the limited binding of a single dimer leads to the same topology change irrespective of whether the substrate is supercoiled (Fig. 4) or relaxed (Fig. 3).
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The SeqA protein has been visualized in fixed cells by immunofluorescence microscopy and in living cells by the expression of SeqA-GFP fusions (Hiraga et al. 1998; Onogi et al. 1999), and was found to localize as discrete foci presumably representing multimers of protein bound to the hemimethylated DNA behind the replication forks. Here, we investigated whether the SeqA4 mutant protein was capable of forming foci. Cells producing wild-type SeqA, SeqA4 at wild-type levels, or SeqA4 at 
25-fold overproduction were grown in glucose-CAA medium to an OD450 of 0.15, fixed, mounted on microscope slides and immunostained using antibody against SeqA (Fossum et al. 2003). The cells containing wild-type SeqA (Fig. 5A) were homogeneous in size and mostly contained two, three or four fluorescent foci in accordance with previous findings (Fossum et al. 2003; Molina & Skarstad 2004). In contrast, cells containing mutant SeqA4 either at wild-type levels or in excess were heterogeneous with different diameters and lengths and did not form discrete foci (Fig. 5A). The immunofluorescence from SeqA4 protein was distributed throughout most of the cells and colocalized with the DNA.
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The seqA4 mutant allele was isolated in a screen for cells deficient in sequestration (von Freiesleben et al. 1994). Such mutants have been shown to re-initiate at already initiated origins and thus replicate asynchronously (von Freiesleben et al. 1994; Lu et al. 1994; Fossum et al. 2003). Here, we investigated whether replication synchrony could be restored by high levels of SeqA4. Cells containing wild-type SeqA, SeqA4 at wild-type levels or SeqA4 at 25-fold overproduction were grown as described previously and then incubated with rifampicin and cephalexin to allow run-out replication and inhibit cell division, respectively, and then fixed and stained with FITC and Hoechst (Lu et al. 1994; von Freiesleben et al. 1994; Torheim et al. 2000; Fossum et al. 2003). Cells with wild-type SeqA contained mainly four chromosomes after replication run-out, which means that they initiated at four origins right before, or at two origins right after cell division. In contrast, cells containing an approximately wild-type level of SeqA4 exhibited asynchronous initiation with peaks at irregular numbers of chromosomes (see Experimental procedures) (Fig. 5B), showing that re-initiation occurs in these cells and that sequestration of new origins does not function. In cells containing a 25-fold excess of mutant SeqA4 protein initiation synchrony was partially restored (Fig. 5B). Most of the cells contained four chromosomes after replication run-out like cells containing wild-type SeqA. Also, the cells with an excess of SeqA4 had an improved growth rate compared to cells with a wild-type level of SeqA4 (the doubling times were 45 and 60 min, respectively, whereas the doubling time for cells expressing normal levels of wild-type SeqA was 27 min).
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Discussion |
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We find here that the SeqA4 protein binds hemimethylated DNA with wild-type affinity but fails to form multimers. This made it possible to identify functions of the SeqA protein that depend on multimerization. Binding of either SeqA or SeqA4 dimers to DNA lead to a topology change equivalent to positive supercoiling. The fact that the effect of the SeqA4 protein was the same as that of the wild-type protein showed that this topology change was caused by binding of single dimers. In contrast, the topology change that resulted in restraint of negative supercoils could not be made by the SeqA4 protein. This shows that for this second type of topology change, binding of single, independent dimers is not sufficient, and formation of a SeqA multimer is required. Recent structure determination of the SeqA N-terminus indicates that it forms a fiber made up of dimers, and structure modelling of the full-length SeqA protein shows that the fiber is likely to be helical (Guarnéet al. 2005). This finding supports the model of the SeqA multimer presented here (Fig. 4C), which is designed to explain the finding that negative supercoils of DNA bound to a SeqA multimer become restrained (Torheim & Skarstad 1999; Klungsøyr & Skarstad 2004). Negatively supercoiled DNA bound to a left-handed SeqA filament will itself adopt a left-handed helical conformation. This will cause the plectonemic supercoils to be redistributed into toroidal turns, and thus be restrained and not recognized by topoisomerase I.
Most of the SeqA protein in the cell seems to be bound to newly formed, hemimethylated DNA at the replication forks and can be seen as foci by immunofluorescence. The SeqA4 protein did not form such foci, showing that specific N-terminus interactions are required for generating such structures. It is probable that the foci consist of helical SeqA filaments bound to hemimethylated DNA. It has been suggested that these structures may be important for localization of replication factories and organization of newly replicated DNA in vivo (Brendler et al. 2000; Norris et al. 2002; Molina & Skarstad 2004).
Origin sequestration is dependent on extensive binding of SeqA to GATC sites
SeqA multimerization does not, however, seem to be essential for origin sequestration because when SeqA4 was over-expressed, the initiation synchrony was partially restored, whereas the mutant protein was still unable to form discrete foci. In contrast, over-expression of the DNA-binding deficient SeqA2 mutant protein did not restore initiation synchrony (Fossum et al. 2003). Thus, it appears that origin sequestration may not require a perfect higher-order structure per se, but does require a high local concentration of SeqA for extensive binding to the hemimethylated GATC sites. At wild-type concentrations of SeqA protein, the higher-order multimer is essential for origin sequestration, probably in order to promote a high local concentration of SeqA. Thus, it is possible that origin sequestration can function independently of chromosome organization as long as a sufficient number of GATC sites are bound by SeqA.
SeqA multimerization is required for normal growth rate in rich medium
Earlier work has shown that even a full asynchrony phenotype because of loss of oriC sequestration does not affect the growth rate significantly (Bach & Skarstad 2004). Thus, the cell cycle seems to flexibly accommodate lack of origin sequestration in such a way that the steady state growth rate is unchanged. Loss of the SeqA protein, on the other hand, leads to significant growth impairment in rich medium (Lu et al. 1994), but only slight overinitiation and no growth impairment during slow growth (Boye et al. 1996). This indicates that the functions of SeqA are most important in rich medium where replication occurs with overlapping replication cycles, but that the loss of function causing slow growth rate is not the loss of sequestration.
The present results show that an excess of SeqA4 partially restores origin sequestration and growth rate. From the arguments mentioned above, however, it is reasonable to assume that the growth improvement is not the result of improved sequestration of oriC. It is more probable that excess SeqA4 also causes partial restoration of one or more of the other functions of SeqA, such as gene regulation, sister chromosome organization, and the building of replication hyperstructures. It is thus also a possibility that at high concentrations, SeqA4 can form partial or "weak" multimers in vivo.
SeqA dimer formation also depends on N-terminal interactions
We find that the mutant SeqA4 protein forms a basic binding unit (a dimer) that binds with wild-type affinity to pairs of hemimethylated GATC sites. Previously it was suggested that the basic binding unit of SeqA is a tetramer (Lee et al. 2001); however, data from structure determination and cross-linking experiments (Lee et al. 2001; Guarnéet al. 2002; Fossum et al. 2003; Guarnéet al. 2005), as well as results obtained with atomic force microscopy (Kang et al. 2005), support the assumption that the basic binding unit of SeqA is a dimer. SeqA protein lacking the N-terminus did not bind to hemimethylated DNA (data not shown; Han et al. 2003), showing that formation of a SeqA dimer depends on N-terminus interactions. We show here that the dimer-forming interactions are present in the SeqA4 N-terminus, and are therefore not dependent on amino acid 25. This amino acid was, as mentioned previously, found to be involved in the formation of higher-order multimers. Recent results by Guarnéet al. (2005) showed that alanine 25 is situated at the hydrophobic interface between neighboring dimers. They found that a mutant protein in which this alanine is substituted with arginine (A25R) was deficient in dimer-dimer aggregation and thus filament formation. It is likely that both this protein and the SeqA4 (A25T) mutant protein studied here have lost the hydrophobic interaction between dimers as they both contain a bulky and hydrophilic amino acid side chain at position 25.
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Experimental procedures |
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All strains used were E. coli K-12 and are listed in Table 1. Cells were grown at 37 °C in Luria-Bertani (LB) medium or AB minimal medium (Clark & Maaløe 1967) supplemented with 1 µg/mL thiamine, 0.2% glucose, 0.5% casamino acids (glucose-CAA medium), and 20 µg/mL tryptophan. Ampicillin 100 µg/mL (Bristol-Myers Squibb) was added when necessary.
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All plasmids used are listed in Table 2. The seqA4 gene was amplified by PCR from the strain UF301. In order to obtain a tag of six histidine residues in the C-terminus of the recombinant protein, plasmids pIO2 (pNEB193 seqA4-His6) and pIO4 (pFH2102 seqA4-His6) were constructed as previously described ((Fossum et al. 2003), see construction of plasmid pSS01). For expression of histidine-tagged SeqA and SeqA4 (from plasmids pSF24 and pIO4, respectively) at approximately wild-type levels (i.e. threefold as measured by Western blot), strains SF55 and IO06 were incubated with 20 and 50 µM IPTG (isopropyl-thio-ß-D-galactoside), respectively, for four generations (to OD4500.15) in AB minimal medium with supplements as described previously (see Bacterial strains and growth conditions). For overproduction of histidine-tagged SeqA4 (25-fold as measured by Western blot) 1 mM IPTG was added. The histidine tag does not affect SeqA function in vivo as histidine-tagged SeqA was able to support initiation synchrony in a 
seqA strain (Fossum et al. 2003). In vitro, a histidine tag does not affect binding of the SeqA protein to DNA (Fossum et al. 2003). Histidine-tagged protein is here referred to by the protein name only.
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Purification of mutant SeqA4 protein
Purification of mutant SeqA4 protein from strain IO06 was performed by HiTrap column affinity chromatography essentially as previously described (Fossum et al. 2003), with the following exception. Instead of dialyzing the SeqA4 containing fractions (eluted from the column) against a buffer containing ammonium chloride (which resulted in precipitation of the mutant protein), the fractions were diluted 1:4 in dilution buffer B (333 mM NaCl, 25 mM Hepes-KOH pH 7.5, 10 mM magnesium acetate, 4 mMß-mercaptoethanol, 20% sucrose, 0.1% Igepal). The SeqA4 protein was further diluted in modified buffer B (500 mM NaCl, 25 mM Hepes-KOH pH 7.5, 10 mM magnesium acetate, 4 mMß-mercaptoethanol, 20% sucrose, 0.1% Igepal) for in vitro studies. Wild-type SeqA protein without histidine tag was purified as described (Skarstad et al. 2000) and diluted in buffer M (300 mM ammonium sulfate, 20 mM Hepes-KOH pH 7.5, 10 mM magnesium acetate, 1 mM EDTA (ethylenediaminetetra-acetic acid), 4 mM DTT (di-thio-threitol), 15% glycerol) for in vitro studies.
Western blotting
Cells were grown to OD4500.15 in AB minimal medium with supplements as described previously (see Bacterial strains and growth conditions) or to OD6000.15 in LB medium. Cells were harvested and SDS (sodium-dodecyl-sulfate) samples were prepared as described (Torheim et al. 2000). SDS samples of purified proteins were prepared as previously described (Fossum et al. 2003). The SDS samples were subjected to 15% or 12% SDS- polyacrylamide gel electrophoresis. Detection of proteins was performed by using rabbit anti-SeqA antibody and ECF Western Blotting Kit (Amersham Biosciences), and quantification was performed using IMAGEQUANT software (Molecular Dynamics) (Torheim et al. 2000).
Gel filtration analysis of full-length protein
Gel filtration analysis was performed as previously described (Fossum et al. 2003).
Limited proteolysis and gel filtration analysis of cleaved protein
Limited proteolysis of wild-type SeqA protein was carried out at 25 °C for 90 min in the presence of 0.094 U trypsin/µg protein (Promega). For the SeqA4 mutant, the reaction was carried out at 20 °C for 90 min in the presence of 0.076 U trypsin/µg protein. Gel filtration analysis of cleaved protein was performed as described (Fossum et al. 2003).
Gel retardation assay
Unmethylated oriC20 fragment was prepared as previously described (Fossum et al. 2003). A 200-bp unmethylated DNA fragment containing one GATC site was amplified from the E. coli chromosome, positions 3926103–3926302 bp (Blattner et al. 1997), using primers 5'-ATA TCG GCC AGG TTC AGT GTG GAG-3' and 5'-ATC AGC ATC GCT TTC ATG CCG-3'. The protocol for preparation of hemimethylated DNA fragments was modified from Kang and coworkers (2003). Unmethylated DNA fragment (2 µg) was added to 50 µL Dam assay solution (Dam methylase buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 5 mMß-mercaptoethanol) and 400 µM S-adenosylmethionine; New England Biolabs Inc.) containing 25 µg SeqA. Dam methylase 40 U (New England Biolabs Inc.) was added and the mixture was incubated at 37 °C for 4 h. After 2 h of the reaction time another 400 µM S-adenosylmethionine was added. The Dam methylase was inactivated at 65 °C for 15 min. The methylation status was checked by cleaving the fragments with the restriction nucleases, DpnI and DpnII (New England Biolabs Inc.). Approximately 50% of the DNA fragments were hemimethylated. For preparation of fully methylated oriC20 fragment, SeqA was omitted from the Dam assay solution and the reaction time was 6 h where another 400 µM S-adenosylmethionine was supplemented after 3 h. Both hemi- and fully methylated fragments were purified by standard phenol/chloroform extraction and ethanol precipitation (Sambrook & Russell 2001). Radioactive labeling of DNA fragments and gel retardation assays were carried out as previously described (Fossum et al. 2003).
Supercoiling assays
The ligase and Topo I assays were performed as previously described (Klungsøyr & Skarstad 2004). Plasmid pBSoriC was used in the Topo I assay in order to compare the results with the results of Torheim and Skarstad (1999). pOC170 was used in the ligase assay. Plasmid pBSoriC is not a suitable substrate for the nicking required in the ligase assay because two of the sites are too close, thus generating a linear molecule.
In a gel without chloroquine, a negatively supercoiled plasmid will move as a compact structure with high mobility, whereas a relaxed, closed plasmid will have lower mobility. In a gel containing a high level of chloroquine, binding of chloroquine will introduce positive supercoils in both types of plasmid. If the plasmid was negatively supercoiled, the negative supercoils will be cancelled and a few positive supercoils introduced; if relaxed, a larger number of positive supercoils will be introduced. A nicked plasmid cannot preserve supercoils and will move with low mobility in both types of gels. To distinguish between positive and negative supercoils, intermediate amounts of chloroquine can be used.
Immunofluorescence microscopy
Cells were prepared for immunofluorescence microscopy and visualized as previously described (Fossum et al. 2003).
Flow cytometry analysis
Preparation of flow cytometry samples was as previously described (Torheim et al. 2000). Flow cytometry analysis was performed using a FACS DiVa Option flow cytometer (Becton Dickinson) equipped with an argon and a krypton laser. Rifampicin and cephalexin treatment yields DNA histograms with integral numbers of chromosomes per cell of value 2n (n = 0, 1, 2, 3, ... ) for cells that initiate synchronously, and DNA histograms with, in addition, irregular numbers of chromosomes (i.e. all values) for cells with asynchronous initiation (Skarstad et al. 1986).
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Acknowledgements |
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This work was supported by the Norwegian Research Council's Basic Science and Functional Genomics Programmes, The Norwegian Cancer Society and the Anders Jahre and Torstedt Foundations.
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Footnotes |
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* Correspondence: E-mail: kirsten.skarstad{at}labmed.uio.no
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References |
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Bach, T. & Skarstad, K. (2004) Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically. Mol. Microbiol. 51, 1589–1600.[CrossRef][Medline]
Bach, T., Krekling, M.A. & Skarstad, K. (2003) Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division. EMBO J. 22, 315–323.[CrossRef][Medline]
Baker, T.A. & Kornberg, A. (1988) Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell 55, 113–123.[CrossRef][Medline]
Bates, D. & Kleckner, N. (2005) Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation. Cell 121, 899–911.[CrossRef][Medline]
Blattner, F.R., Plunkett, G.
III, Bloch, C.A., et al. (1997) The complete genome sequence of Escherichia coli K-12. Science
277, 1453–1462.
Boye, E., Stokke, T., Kleckner, N. & Skarstad, K. (1996) Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins. Proc. Natl. Acad. Sci. USA
93, 12206–12211.
Brendler, T. & Austin, S. (1999) Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences. EMBO J. 18, 2304–2310.[CrossRef][Medline]
Brendler, T., Sawitzke, J., Sergueev, K. & Austin, S. (2000) A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation. EMBO J. 19, 6249–6258.[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, 967–979.[CrossRef][Medline]
Clark, D.J. & Maaløe, O. (1967) DNA replication and the division cycle in Escherichia coli. J. Bacteriol. 181, 2683–2688.
Fossum, S., Søreide, S. & Skarstad, K. (2003) Lack of SeqA focus formation, specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2. Mol. Microbiol. 47, 619–632.[CrossRef][Medline]
Fujikawa, N., Kurumizaka, H., Yamazoe, M., Hiraga, S. & Yokoyama, S. (2003) Identification of functional domains of the Escherichia coli SeqA protein. Biochem. Biophys. Res. Commun. 300, 699–705.[CrossRef][Medline]
Guarné, A., Zhao, Q., Ghirlando, R. & Yang, W. (2002) Insights into negative modulation of E. coli replication initiation from the structure of SeqA-hemimethylated DNA complex. Nature Struct. Biol. 9, 839–843.[Medline]
Guarné, A., Brendler, T., Zhao, Q. et al. (2005) Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization. EMBO J. 24, 1502–1511.[CrossRef][Medline]
Han, J.S., Kang, S., Lee, H., Kim, H.K. & Hwang, D.S. (2003) Sequential binding of SeqA to paired hemi-methylated GATC sequences mediates formation of higher order complexes. J. Biol. Chem.
278, 34983–34989.
Han, J.S., Kang, S., Kim, S.H., Ko, M.J. & Hwang, D.S. (2004) Binding of SeqA protein to hemi-methylated GATC sequences enhances their interaction and aggregation properties. J. Biol. Chem.
279, 30236–30243.
Hansen, F.G. & von Meyenburg, K. (1979) Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda tnA. Mol. Gen. Genet. 175, 135–144.[CrossRef][Medline]
Hiraga, S., Ichinose, C., Niki, H. & Yamazoe, M. (1998) Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA-protein complexes in E. coli. Mol. Cell 1, 381–387.[CrossRef][Medline]
Hiraga, S., Ichinose, C., Onogi, T., Niki, H. & Yamazoe, M. (2000) Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli. Genes Cells 5, 327–341.[Abstract]
Kang, S., Han, J.S., Park, J.H., Skarstad, K. & Hwang, D.S. (2003) SeqA protein stimulates the relaxing and decatenating activities of topoisomerase IV. J. Biol. Chem.
278, 48779–48785.
Kang, S., Han, J.S., Kim, K.P., et al. (2005) Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences. Nucleic Acids Res.
33, 1524–1531.
Klungsøyr, H.K. & Skarstad, K. (2004) Positive supercoiling is generated in the presence of Escherichia coli SeqA protein. Mol. Microbiol. 54, 123–131.[CrossRef][Medline]
Lee, H., Kang, S., Bae, S.H., Choi, B.S. & Hwang, D.S. (2001) SeqA protein aggregation is necessary for SeqA function. J. Biol. Chem.
276, 34600–34606.
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]
Molina, F. & Skarstad, K. (2004) Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism. Mol. Microbiol. 52, 1597–1612.[CrossRef][Medline]
Norris, V., Demarty, M., Raine, D., Cabin-Flaman, A. & Le Sceller, L. (2002) Hypothesis: hyperstructures regulate initiation in Escherichia coli and other bacteria. Biochimie 84, 341–347.[Medline]
Onogi, T., Niki, H., Yamazoe, M. & Hiraga, S. (1999) The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli. Mol. Microbiol. 31, 1775–1782.[CrossRef][Medline]
Roth, A. & Messer, W. (1995) The DNA binding domain of the initiator protein DnaA. EMBO J. 14, 2106–2111.[Medline]
Russell, D.W. & Zinder, N.D. (1987) Hemimethylation prevents DNA replication in E. coli. Cell 50, 1071–1079.[CrossRef][Medline]
Sambrook, J. & Russell, D.W. (2001) Molecular Cloning. A Laboratory Manual, 3rd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Skarstad, K., Boye, E. & Steen, H.B. (1986) Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J. 5, 1711–1717.[Medline]
Skarstad, K., Lueder, G., Lurz, R., Speck, C. & Messer, W. (2000) The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated Oric. Mol. Microbiol. 36, 1319–1326.[CrossRef][Medline]
Slater, S., Wold, S., Lu, M. et al. (1995) E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82, 927–936.[CrossRef][Medline]
Torheim, N.K. & Skarstad, K. (1999) Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at Oric. EMBO J. 18, 4882–4888.[CrossRef][Medline]
Torheim, N.K., Boye, E., Løbner-Olesen, A., Stokke, T. & Skarstad, K. (2000) The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein. Mol. Microbiol. 37, 629–638.[CrossRef][Medline]
von Freiesleben, U., Krekling, M.A., Hansen, F.G. & Lobner-Olesen, A. (2000) The eclipse period of Escherichia coli. EMBO J. 19, 6240–6248.[CrossRef][Medline]
von Freiesleben, U., Rasmussen, K.V. & Schaechter, M. (1994) SeqA limits DnaA activity in replication from oriC in Escherichia coli. Mol. Microbiol. 14, 763–772.[Medline]
Weitao, T., Nordstrom, K. & Dasgupta, S. (1999) Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure. Mol. Microbiol. 34, 157–168.[CrossRef][Medline]
Weitao, T., Nordstrom, K. & Dasgupta, S. (2000) Escherichia coli cell cycle control genes affect chromosome superhelicity. EMBO Rep. 1, 494–499.[CrossRef][Medline]
Received: 12 May 2005
Accepted: 19 July 2005
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-32P]-ATP labeled DNA fragments (0.35 nM) in the presence of competitor DNA (4 µg) at 37 °C for 30 min. Lane 1: no protein; lanes 2–8: 1, 5, 10, 25, 50, 100, 250 nM SeqA; lanes 9–15: 1, 5, 10, 25, 50, 100, 250 nM SeqA4. The samples were run on a 5% nondenaturing polyacrylamide gel. The different complexes formed are indicated (I–IV and M). (B) The percentage of free DNA fragment is plotted as a function of protein concentration (
, SeqA; 
, SeqA4).
