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Originally published In Press as doi:10.1074/jbc.M101339200 on July 16, 2001

J. Biol. Chem., Vol. 276, Issue 37, 34600-34606, September 14, 2001
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SeqA Protein Aggregation Is Necessary for SeqA Function*

Ho LeeDagger §, Sukhyun KangDagger §, Sung-Hun Bae||, Byong-Seok Choi||, and Deog Su HwangDagger **

From the Dagger  Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea and the || Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea

Received for publication, February 12, 2001, and in revised form, July 10, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Escherichia coli, initiation of chromosomal replication occurs at the origin of chromosomal replication, oriC (1, 2). Within the oriC DNA region are 11 GATC sequences. Like all chromosomal GATC sites, these sequences are methylated by Dam methyltransferase at the 6-amino group of the adenine residue (3). Following replication, GATC sites are in a hemimethylated state; the parent strand retains its methylation while the nascent strand lacks methylation (4). Conversion of DNA from the hemi to fully methylated state is a relatively rapid process. However, at oriC, methylation is inhibited for one-third of the cell cycle. This delay is due to SeqA protein sequestration of hemimethylated oriC; sequestration is important in preventing premature initiation (5, 6, 18). Preferential binding of SeqA to hemimethylated DNA requires at least two hemimethylated GATC sequences situated on the same face of the DNA helix (7, 8). Therefore, it was proposed that stable binding of SeqA to hemimethylated DNA requires cooperative interaction between SeqA proteins.

Given the role of SeqA in oriC sequestration, it has been suggested that SeqA is a negative modulator of chromosomal initiation (5, 9). In support of this hypothesis, seqA mutants demonstrate asynchronous and increased frequencies of initiation. In addition to this regulatory role, SeqA appears to be involved in nucleoid organization and chromosomal segregation (10, 11, 30, 31). Even in the absence of oriC, SeqA forms foci on chromosomal DNA. These foci are dispersed in dam mutants and depend upon continuous chromosomal replication, implying that SeqA foci formation is dependent upon hemimethylated chromosomal DNA (8, 10). Cooperative interaction and aggregation of SeqA on hemimethylated DNA templates (6-8, 12) along with formation of SeqA foci on the chromosome (10, 11) indicate that interactions must occur between SeqA proteins. In this report, we analyzed the physical interaction between SeqA tetramers and found that amino acid 9, a glutamic acid residue, is required for proper SeqA interaction.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Sources were as follows: restriction enzyme and cloning enzymes, Promega; dissucinimidyl suberate, Pierce; MacConkey Agar, Difco; QuikChangeTM site-directed mutagenesis kit, Strategene. Unless otherwise indicated, other reagents were purchased from Sigma.

Bacterial Strains and Plasmid DNAs-- The E. coli WBT13 (W3110 iciA::KmR) and NK9050 (seqA::TcR) (5) were used for preparation of wild-type and mutant SeqA protein, respectively. E. coli DHP1 (F- Delta cya glnV44(AS) recA1 endA1 gyrA96(Nalr) thi1 hsdR17 SpoT1 rfbD1) was used in the bacterial two-hybrid system (13). E. coli DH5alpha (14) and GM3819 (dam16::KmR) (15) were used for preparations of fully methylated and unmethylated plasmid DNAs, respectively. The plasmids pT25 (13), pT18 (13), pBAD18 (16), and pBMA1 (17) are previously described. The seqA gene was inserted into EcoRI-HindIII site of pBAD18, constructing pBAD18-seqA.

Protein Preparation-- SeqA protein was purified from the SeqA overproducing strain, BL21(pLys, pSS1) as previously described (18). One unit of SeqA activity was defined as the activity required to convert one-quarter of input hemimethylated DNA (1.5 fmol) to slowly migrating complex in gel shift assays (0.375 fmol).

Preparation of partially purified wild-type SeqA from the SeqA non-overproducing strain E. coli WBT13 was performed as previously described (19). The KCl extract of the membrane fraction from lysed cells was loaded onto a heparin-agarose column. Active column fractions were pooled and (NH4)2SO4 was added to 1.5 M. The mixture was applied to a phenyl-Sepharose CL-4B (Amersham Pharmacia Biotech) column equilibrated in buffer A (25 mM HEPES (pH 7.8), 0.1 mM EDTA, 1 mM DTT,1 and 15% glycerol) containing 1.5 M (NH4)2SO4. The column was then washed with 1.5 M (NH4)2SO4 in buffer A, followed by a further wash with 50 mM KCl in buffer A. SeqA was eluted using buffer A containing 50% ethylene glycol. The active fractions were pooled, dialyzed in buffer A containing 50 mM KCl, then applied to a Fast-flow S Sepharose (Sigma) column equilibrated in buffer A containing 50 mM KCl. SeqA was eluted using a linear gradient ranging from 50 mM to 1 M KCl in buffer A. The active fractions were pooled (fraction IV, 0.2 mg/ml) and used in further experiments.

Crude fractions of wild-type and mutant SeqA proteins (fraction A) were prepared as previously described (19) with minor modifications. E. coli NK9050 cells harboring the indicated seqA on pBAD18 were grown in LB medium to an optical density at 600 nm of 0.1 to 0.2, followed by the addition of L-(+)-arabinose to a concentration of 0.2%. 6 h after induction, cells were harvested and resuspended in 25 mM HEPES (pH 7.8), 1 mM EDTA, and 1 mM dithiothreitol. Cell lysis and KCl extraction were performed, as previously described (19), to obtain fraction A.

Gel-shift Assay-- Gel-shift assays using a 75-mer synthetic hemimethylated DNA containing four GATC sequences, unless indicated, was performed as previously described (18).

Chemical Cross-linking-- The indicated protein was incubated with 7 or 20 mM of the covalent cross-linker, dissucinimidyl suberate (DSS, Pierce), at 4 °C for the indicated times (20). Free reactive groups were quenched with 50 mM Tris (pH 7.5). Quenched reaction mixtures were subjected to 12% SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue or silver staining.

Construction and Expression of the Mutant seqA-- Experiments using the bacterial two-hybrid system were performed as previously described (13). The open reading frame of the seqA gene was inserted into the PstI-KpnI site of pT25 and KpnI-HindIII site of pT18 using PCR, constructing pT25-seqA and pT18-seqA, respectively.

To introduce random mutations on seqA, mutagenic PCR was performed as previously described (21) with minor modifications. The seqA open reading frame in the pBAD18-seqA was amplified using the oligonucleotides, 5'-TGGACTGCAGGGAAAACGATTGAAGTTGATGA-3' and 5'-GGGGGGGTACCTTAGATAGTTCCGCAAACC-3' (PstI and KpnI restriction sites are in bold), as primers for 30 cycles of PCR (94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min). PCR products were subjected to agarose gel electrophoresis and purification, restricted with PstI and KpnI, and ligated into the multicloning site of pT25. The resulting DNAs were transformed into E. coli DHP1 cells harboring pT18-seqA. Transformed cells were spread on MacConkey agar (Difco) plates containing 1% maltose, 50 µg/ml ampicillin, 17 µg/ml chloramphenicol, and 10 µg/ml phosphomycin, and incubated 15 to 21 h at 30 °C. Following the incubation, paler red colonies were inoculated into 2 ml of LB medium containing 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside and antibiotics as described above, and cultured at 30 °C for 12 h. 100 µl of culture was used to measure beta -galactosidase activity as previously described (22). The plasmid pT25-seqA was isolated from clones whose beta -galactosidase activities were lower than that of DHP1 cells harboring pT25-seqA and pT18-seqA. Mutated sequences of the seqA gene were identified by DNA sequencing.

To obtain deletion mutants of the seqA in pBAD18-seqA, the primers 5'-GGATGGTACCTAAAACGATTGAAGTTGATGATG-3' and 5'-TTTTGGTACCTTACGCTTCGACGATAGCAGG-3'; 5'-TCGTGGTACCTAAGCCGGTCAAAACGATTAAA-3' and 5'-GCAGGGTACCTTAATCTGCCGCAAAGTAAACGC-3'; and 5'-TTGCGGTACCTGAACAAACGCTGCTGAAAAAT-3' and 5'-GGGGGGGTACCTTAGATA GTTCCGCAAACC-3' (KpnI restriction sites are in bold) were used for PCR, restricted with KpnI, and ligated into pT25, thereby constructing pT25-seqA-(1-59), pT25-seqA(60-124), and pT25-seqA(125-181), respectively.

To obtain overproducing mutant SeqA clones, the indicated substitutions were introduced into pBAD18-seqA using the QuikChangeTM site-directed mutagenesis kit (Strategene). Mutated plasmids were then transformed into E. coli NK9050 to avoid the contamination of purified wild-type SeqA. Also, mutated seqA genes were subcloned into pT25 using PCR to detect any interaction with pT18-seqA using the bacterial two-hybrid system.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Concentration-dependent Reversible Aggregation of SeqA-- Both in vitro and in vivo data suggest a physical interaction between SeqA proteins (6, 10-12). Therefore, we wanted to examine SeqA-SeqA interactions and determine which amino acids are required for this association. To determine the number of SeqA proteins in a SeqA-SeqA complex, we employed Superose 12 gel-filtration chromatography (Fig. 1). Low levels of SeqA loaded onto the column eluted in later fractions. As the amount of purified SeqA protein loaded onto the column increased, SeqA proteins eluted in earlier fractions, implying that these fractions contain higher molecular weight forms of SeqA protein (Fig. 1, A and B). For example, loading 0.2 and 1 µg of SeqA protein onto the column resulted in elution of SeqA in 90-kDa complexes, while loading 5 and 40 µg resulted in elution of SeqA in 200- and 360-kDa complexes, respectively. Loading 2.5 mg of SeqA caused elution of SeqA near the void volume (data not shown). The activity profiles of SeqA eluted from the Superose 12 column paralleled protein levels as detected by SDS-polyacrylamide gel electrophoresis. Even though SeqA-SeqA complexes eluted at different fractions dependent upon SeqA protein concentration, the specific activities of SeqA in each peak fraction were similar to each other (Table I). Since differing amounts of purified SeqA protein (all from the same purification fraction) were analyzed using the same gel-filtration column, the similar specific activities of each peak indicate that SeqA reversibly aggregates or multimerizes depending upon concentration. The broad elution patterns of SeqA in various molecular weight forms also suggests that SeqA exists as a mixture of heterogeneously aggregated forms at a given concentration.


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  		Fig. 1.   SeqA aggregates in a concentration-dependent manner. A, the indicated amounts of purified SeqA (1.3 mg/ml) were resuspended in 50 µl of 1 M KCl in buffer A (25 mM HEPES (pH 7.8), 0.1 mM EDTA, 1 mM dithiothreitol, and 15% glycerol), loaded onto a Superose 12 PC 3.2/30 SMART gel-filtration column, and eluted with buffer A containing 1 M KCl. 40 µl of each fraction was collected and SeqA activity determined using gel-shift assays as described under "Experimental Procedures." Apoferritin (horse spleen, 443 kDa), beta -amylase (sweet potato, 200 kDa), alcohol dehydrogenase (yeast, 150 kDa), and albumin (bovine serum, 66 kDa) were used as molecular weight markers. B, the indicated column fractions were analyzed using 14% SDS-polyacrylamide gel electrophoresis, and visualized by silver staining. Carbonic anhydrase (29 kDa) and trypsinogen (24 kDa) were used as molecular mass markers.

                              
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Table I
Reversible aggregation of SeqA
The peak fractions described in Figs. 1A and 2A were compared. The protein amounts in each fraction were determined using Western blot analysis as compared to known amounts of SeqA protein.

Tetrameric Behavior of SeqA Protein-- Although the seqA nucleotide sequence predicts a 20-kDa polypeptide (5), we found that low levels of SeqA loaded onto a Superose 12 gel-filtration column eluted as a 90-kDa peak (Fig. 1). Using the same gel-filtration column, we loaded similar amounts of SeqA protein partially purified from a non-overproducing, wild-type strain. SeqA activity still eluted at the 90-kDa peak and the specific activity of this SeqA was similar to that of SeqA purified from the overproducing strain (Table I). These results indicate that SeqA protein purified from an overproducing strain is identical to SeqA protein purified from a non-overproducing strain.

To further study SeqA-SeqA interactions, multimerization and aggregation was also analyzed using glycerol gradient sedimentation (Fig. 2C). Low levels of SeqA protein sedimented in later fractions, indicating that these fractions contain lower molecular weight forms of SeqA. Increasing amounts of SeqA protein resulted in sedimentation of SeqA in earlier fractions containing larger molecular weight forms of SeqA. These results are in agreement with the gel-filtration experiments (Fig. 1). The sedimentation coefficient of the SeqA peak using 5 µg of protein was determined to be 4.3 × 10-13 (Fig. 2D). The Stokes radius of SeqA eluted from the Superose 12 gel-filtration column in the 90-kDa peak was calculated as 4.2 nm (Fig. 2B). These two values indicate that SeqA has a molecular mass of 77 kDa with an axial ratio of 9 as prolate ellipsoid or 10.5 as oblate ellipsoid (23, 24). Therefore, at concentrations of 11-12 µg/ml (Table I), SeqA exists as a homotetramer composed of 20-kDa polypeptides. These tetramer units reversibly aggregate in a concentration-dependent manner.


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  		Fig. 2.   Tetrameric nature of non-overproduced SeqA. A, 100 µg of fraction IV, obtained from SeqA nonoverproducing WBT13 cells, was precipitated by ammonium sulfate and resuspended in 50 µl of buffer A containing 1 M KCl, followed by loading onto Superose 12 column as described in the legend to Fig. 1A. Or, 10 µg of fraction IV without ammonium sulfate precipitation was loaded onto the Superose 12 column. B, based on the values obtained from panel A, the Stokes radius of SeqA was determined as described previously (23, 24). C, the indicated amounts of purified SeqA were loaded onto 4.5 ml of a 18-38% (v/v) glycerol gradient in buffer A containing 1 M KCl. The gradients were centrifuged for 25 h at 48,000 rpm in a Beckman SW 50.1 rotor. Fractions (110 µl each) were collected from the bottom. D, the sedimentation coefficient of SeqA was determined using the value obtained from the peak fraction of 5 µg of loading in panel C as previously described (23, 24). Catalase (238 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) were used as molecular weight markers.

The tetrameric property of SeqA was further studied using cross-linking analysis. In these experiments, the cross-linking agent, DSS, was used. The homobifunctional N-hydroxysuccinimide ester of DSS cross-links close proximity to amine groups and generates non-dissociable conjugated polypeptides. Although the reactivity of DSS is variable from protein to protein, the cross-linking of SeqA was comparable to E. coli SSB and RcsB, which are both homotetramers composed of 18.8-kDa polypeptides (25) and 24-kDa monomers,2 respectively (Fig. 3). Incubation of SSB with 7 mM DSS for 2 h produced protein bands corresponding to the molecular weights of monomer, dimer, trimer, and tetramer forms as detected using SDS-polyacrylamide gel electrophoresis (Fig. 3A, lane 2). Under the same conditions, monomeric and dimeric SeqA bands with minor trimeric and tetrameric bands were observed (Fig. 3B, lane 2). Incubation of SeqA with a higher concentration of DSS, 20 mM, increased levels of trimeric and tetrameric SeqA. However, at the same concentration of DSS, tetrameric SSB and monomeric RcsB were the most predominant forms detected (Fig. 3, A, lane 3, and C), indicating that the intermolecular cross-linking occurred insignificantly. These results suggest that two of the four subunits in a SeqA tetramer are closer to each other than the other two and are therefore easily conjugated at lower DSS concentrations. At higher DSS levels (20 mM), SeqA forms larger than the tetramer were seen; these higher molecular weight forms were not observable in SSB nor RcsB. These larger SeqA forms were probably produced by aggregation of tetrameric SeqA.


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  		Fig. 3.   Chemical cross-linking of SeqA proteins. DSS cross-linking of the indicated proteins were performed as described under "Experimental Procedures" and analyzed using 12% polyacrylamide gel electrophoresis. Proteins were visualized by Coomassie Blue (A) or silver staining (B and C). Molecular weight markers (Sigma) were beta -galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), albumin (egg, 45 kDa), glycerol-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), and trypsin inhibitor (20 kDa).

The N Terminus of SeqA Participates in the SeqA-SeqA Interaction-- A bacterial two-hybrid system was developed to study SeqA-SeqA interactions based upon the functional complementation of the N- (T25) and C- (T18) terminal regions of Bortdetella pertussis adenylate cyclase (13). Interaction of two proteins or protein domains fused to T25 and T18 results in the reconstitution of active adenylate cyclase, and therefore synthesis of cAMP. The cAMP catabolically activates the reporter lacZ gene in a cya mutant of E. coli.

Open reading frames of the genes indicated in Table II were cloned into the appropriate sites of plasmids pT25 and pT18, which express T25 and T18, respectively. As a positive control, pT25-zip/pT18-zip, which contains the sequence for a 35-amino acid leucine zipper motif of GCN4 fused to both pT25 and pT18 (13), was tested for interaction using the bacterial two-hybrid system. Under our assay conditions, pT25-zip/pT18-zip exhibited 3-fold more beta -galactosidase activity than pT25/pT18 alone. Variable combinations of the genes for E. coli proteins DnaA, Skp, and trigger factor did not show significant beta -galactosidase activities. However, pT25-seqA/pT18-seqA, a construct containing the entire seqA open-reading frame in both pT25 and pT18, had 5-fold more beta -galactosidase activity than pT25/pT18. These results also support a functional SeqA-SeqA interaction, corroborating gel-filtration and glycerol gradient data.

                              
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Table II
SeqA-SeqA interaction in a bacterial two-hybrid system
The open reading frames of zip, seqA, dnaA, hlpA, and tif were inserted into plasmids pT25 and pT18, then transformed into E. coli DHP1. The transformed cells harboring both of the pT18 and pT25 derivatives were grown in LB medium containing 0.2 mM IPTG, 50 µg/ml ampicillin, 17 µg/ml chloramphenicol, and 10 µg/ml phosphomycin. After incubation at 37 °C for 12 h, beta -galactosidase activities in 100 µl of each culture were measured, compared to the beta -galactosidase activity of the cells harboring pT25 and pT18, then indicated as the folds of activities. The values obtained from three independent experiments were statistically described.

To identify amino acids or domains involved in SeqA-SeqA interactions, random mutations were introduced into the seqA gene on pT25-seqA using mutagenic PCR. Clones exhibiting reduced beta -galactosidase activities were identified and sequenced (Table III). Among the identified 19 point mutations, 7 were within amino acids 9 through 24. Because of the high number of mutations in this region, participation of the N terminus in SeqA-SeqA interactions was examined (Fig. 4A). The open reading frame of seqA was divided into three parts and each sequence was cloned into pT25. Although pT25-seqA-(1-59), the construct containing the N-terminal region of SeqA, showed less beta -galactosidase activity than pT25-seqA, its beta -galactosidase activity was significant when compared with clones containing the middle or C-terminal region of SeqA.

                              
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Table III
Mutants defective in SeqA-SeqA interaction
pT25-seqA mutant plasmids were transformed into cells containing pT18-seqA and analyzed as described under "Methods and Materials." The beta -galactosidase activities of cells harboring pT25-seqA or its mutants were determined, compared to those of cells harboring pT25, and described as relative activity. Mutated amino acids were described as the single-letter code of amino acids and numbers commencing at the N-terminal end.


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  		Fig. 4.   The N-terminal region is responsible for SeqA-SeqA interactions. Interaction of wild-type SeqA, expressed from pT18-seqA, with mutant SeqA, expressed from pT25-seqA derivatives, containing the indicated domains (A) or amino acid substitutions (B) were measured using the bacterial two-hybrid system as described under "Experimental Procedures." Cells harboring pT18-seqA and the indicated plasmids were grown in 2 ml of LB medium containing 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside at 37 °C for 12 h. 100 µl of each culture was used to measure beta -galactosidase activities. Results were indicated as Miller units as previously described (22).

Several seqA mutants exhibiting reduced beta -galactosidase activity were isolated and sequenced. Every mutant contained multiple amino acid changes. Therefore, mutations expressing single amino acid substitutions were introduced into pT25-seqA using site-directed mutagenesis and the resultant mutants tested using the bacterial two-hybrid system (Fig. 4B). Among those tested, SeqA(E9K), in which the 9th amino acid reside glutamic acid was mutated to lysine, exhibited the most reduced beta -galactosidase activity. These results indicate that the N-terminal region of SeqA protein, especially Glu9, participates in SeqA-SeqA interactions.

Altered Binding of SeqA(E9K) to the Hemimethylated DNA-- SeqA mutant proteins as described in Fig. 4B were obtained as follows: first, the open reading frame of each seqA mutant was cloned into pBAD18, an arabinose inducible expression vector (16). Clones were expressed in the seqA deletion mutant, NK9050 (seqA::TcR), a seqA null strain, to avoid wild-type protein contamination. Activities of crudely purified SeqA and SeqA mutants (fraction A) were determined by gel-shift assays using a synthetic, 71-mer, hemimethylated DNA containing 3 GATC sequences (7). Purified and crude wild-type SeqA bound identically to the hemimethylated DNA (Fig. 5). SeqA(E9K) did not bind the probe, while bound SeqA(K68E) migrated faster than bound wild-type. Binding of other SeqA mutants to the hemimethylated DNA probe was similar to that of the wild-type protein. Western blot analyses of those crudely purified fractions showed that amounts of the expressed proteins were similar and their molecular weights were identical (data not shown).


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  		Fig. 5.   SeqA(E9K) is defective in binding hemimethylated DNA. Cells harboring the SeqA wild type (indicated as Wt) or indicated mutant overproducing plasmid were used to prepare fraction A. SeqA activities were measured in gel-shift assays using a synthetic 71-mer, hemimethylated DNA containing three GATC sequences (7) as described under "Experimental Procedures." A, lane 1, no protein; lanes 2 and 3, 40 and 80 ng of purified SeqA (as SeqA), respectively; lanes 4 and 5, 0.45 and 0.9 µg of wild type SeqA fraction A, respectively; lanes 6 and 7, 0.5 and 1.0 µg of SeqA(E9K) fraction A, respectively. B, lane 1, no protein; lane 2, 0.9 µg of wild-type fraction A; lanes 3-14, 0.45 and 1.8 µg of each indicated mutant fraction A.

The hemimethylated DNA probe used in the above assays possessed randomly chosen GATC sites (7). To study SeqA interactions with oriC, a hemimethylated DNA possessing the AT-rich, 13-mer region of oriC containing either 6 (Fig. 6A) or 4 (Fig. 6, B and C) GATC sites (18) was used. SeqA(E9K) was able to bind these hemimethylated DNAs, but with an altered binding pattern as compared with wild-type. While the wild-type protein exhibited several distinct band shifts, SeqA(E9K) only yielded one shifted band (Fig. 6A). This band migrated slightly faster than the upper band shifted by wild-type. To ensure that the single band shifted by SeqA(E9K) was in fact due to SeqA binding, gel-shift assays of SeqA(E9K) were performed using un- and fully methylated probe (Fig. 6B). SeqA was unable to bind either template. In addition, SeqA(E9K) bound to hemimethylated DNA was supershifted by SeqA antiserum (Fig. 6C). These results indicated that hemimethylated DNA binding was due to SeqA(E9K). The wild-type SeqA and SeqA(E9K)-hemimethylated DNA complexes separated in Fig. 6A were analyzed using the OP-Cu(II) in situ footprinting assays (Fig. 6D). The binding patterns of wild-type protein were identical to the patterns reported previously (18). The similar binding pattern of SeqA(E9K) complex to that of wild-type slow migrating complex indicates that SeqA(E9K) bound to the four GATC sequences.


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  		Fig. 6.   Altered binding of SeqA(E9K) to the hemimetylated oriC. Purified SeqA and fraction As of wild type SeqA and SeqA(E9K) are described as SeqA, Wt, and E9K, respectively. A, gel-shift assays were performed using the hemimethylated EcoRI-HindIII fragment, containing oriC and 6 GATC sequences, isolated from pBMA1 as previously described (17, 18). Lane 1, no protein; lanes 2-7, 0.06, 0.12, 0.24, 0.48, 0.95, and 1.9 µg of wild type fraction A, respectively; lanes 8-13, 0.06, 0.12, 0.24, 0.48, 0.95, and 1.9 µg of SeqA(E9K) fraction A, respectively. B, unmethylated, fully methylated, and hemimethylated synthetic, 75-mer DNAs containing the 13-mers were prepared as previously described (18). The indicated proteins were 20 and 40 ng of purified SeqA, 0.45 and 0.9 µg of wild-type fraction A, or 0.45 and 0.9 µg of SeqA(E9K) fraction A. C, gel-shift assays using a synthetic 75-mer hemimethylated DNA, containing 4 GATC sequences, of the 13-mer region of oriC ("Experimental Procedures" and Ref. 18) were performed with 40 ng of purified SeqA (lanes 2-4), 0.45 µg of wild-type fraction A (lanes 5-7), or 0.45 µg of SeqA(E9K) fraction A (lanes 8-10). Pre-Ab and Ab-SeqA indicate preimmune serum and SeqA antiserum, respectively. D, free DNA (Free), fast (Fast), and slow (Slow) migrating complexes of wild-type SeqA, and SeqA(E9K) (E9K) complex foot;4310f1;10;ZPICKFOOT;Fn1shown in the panel A were analyzed using OP-Cu(II) in situ footprinting analysis as described previously (18). L, M, and R indicate 13-mer, L, M, R, respectively. GATC sequences are denoted as boxes. G and C indicate Maxam and Gilbert sequencing reactions of G and C, respectively.

To further study SeqA(E9K), a partially purified fraction of the mutant was used in Superose 12 gel-filtration chromatography. SeqA(E9K) activity, which was specific to hemimethylated DNA, eluted as a 90-kDa peak, similar to the wild-type protein (Fig. 7A), indicating that SeqA(E9K) also exists as tetramers. However, unlike wild-type, the SeqA(E9K) protein patterns as determined by SDS-polyacrylamide gel electrophoresis were not in parallel with its activity (Fig. 7B). While the peak of SeqA(E9K) activity was found in fraction 18, the protein peak was in fraction 16. These results indicate that SeqA(E9K) aggregate is either less active than the tetramer or inactive. In contrast to wild-type SeqA, SeqA(E9K) tetramer irreversibly aggregates and loses its activity. Concentration of SeqA(E9K) through purification increased aggregation, resulting in poor recovery of SeqA(E9K) activity and a failure to obtain homogeneous SeqA(E9K) (data not shown).


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  		Fig. 7.   SeqA(E9K) tetramer aggregate is inactive. SeqA(E9K) was overproduced using NK9050(seqA::TcR) (5), and crudely purified as described under "Experimental Procedures." To the resuspended (NH4)2SO4 precipitate of PEI supernatant, (NH4)2SO4 was added to a final concentration of 1.5 M, then loaded onto a phenyl-Sepharose CL-4B column equilibrated with buffer A containing 1.5 M (NH4)2SO4 and 50 mM KCl. The column was washed with 1.5 M (NH4)2SO4 in buffer A followed by further washing with 50 mM KCl in buffer A. SeqA(E9K) was eluted with buffer A containing 50% ethylene glycol. A, 50 µl of the SeqA(E9K) phenyl-Sepharose fraction or 1 µg of purified SeqA was analyzed using Superose 12 gel-filtration column as described in the legend to Fig. 1. B, the indicated column fractions of SeqA(E9K) were subjected to a 14% SDS-polyacrylamide gel electrophoresis followed by visualization using silver staining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Assuming a cylindrical shape of E. coli with a diameter and length of 1 and 2 µm, respectively, 1,000 molecules of SeqA (5) is roughly equivalent to a concentration of 20 µg/ml (or 1 µM). Given the tetrameric nature of SeqA at concentrations of 11 to 12 µg/ml in vitro (Figs. 1 and 2, and Table I), it is likely that SeqA associates as a tetramer in vivo. The existence of higher molecular weight forms of SeqA, all possessing similar specific activities (Fig. 1 and Table I), indicates that SeqA tetramers reversibly multimerize or aggregate in a concentration-dependent manner. Broad elution and sedimentation patterns of SeqA in gel-filtration chromatography and glycerol gradients suggest that SeqA tetramers form a heterogeneously aggregated mixture at a given concentration.

In previous studies it was determined that SeqA requires two hemimethylated GATC sequences, spaced up to 31 bases apart, on the same face of the DNA helix, for stable binding (7, 8). It was proposed that the binding was stabilized by cooperative interaction between SeqA proteins transiently bound to each GATC site. In the wild-type SeqA fast migrating complex shown in Fig. 6D and Ref. 18, it appears that one SeqA tetramer binds to the GATC site in the L 13-mer; another SeqA appears to bind to one of the other 3 GATC sites. Once bound the two SeqA proteins interact with each other, thus forming the fast-migrating complex. In this case, binding to the other three sites is distributive, and then those bindings could be undetected in in situ footprinting analysis. In this and previous reports, SeqA binding experiments were performed using more or less than 100 nM (or 2 µg/ml) SeqA (6-8, 18). Therefore, the observed concentration-dependent aggregates formed prior to DNA binding would not directly contribute to the hemimethylated DNA binding at lower SeqA concentrations. Rather, the close proximity of SeqA tetramers transiently bound to GATC sequences would allow for tetramer interaction, to stabilize hemimethylated DNA binding as previously proposed (7, 8), which would be similar in manner to the concentration-dependent aggregation of the SeqA protein. Corroborating this proposal, large complexes of SeqA bound to hemimethylated oriC, containing more than 11 GATC sequences, can be observed using electron microscopy (12) and gel-shift experiments (6).

Of all the single substitution seqA mutations tested for SeqA-SeqA interactions using the two-hybrid assay, the SeqA(E9K) mutant exhibited the lowest interaction. However, all the mutants containing multiple amino acid substitutions exhibited less SeqA-SeqA interactions than the SeqA(E9K) mutant (Fig. 4B and Table III). These results indicate that other amino acids contribute, either synergistically or additively, to proper SeqA-SeqA interactions. The two-hybrid system used in screening for seqA mutants defective in SeqA-SeqA interaction cannot discriminate between the tetramer and oligomer form of SeqA tetramers. It appears that the SeqA(E9K) mutant is defective in the oligomerization. Gel-filtration studies (Fig. 7) demonstrated that SeqA(E9K), like wild-type SeqA, forms tetramers. However, unlike wild-type, SeqA(E9K) is unable to form active aggregates. SeqA(E9K) also exhibits altered gel-shift binding patterns as compared with wild-type when probed with a hemimethylated, 6 or 4 GATC sequence-containing DNA oligonucleotide (Fig. 6). SeqA(E9K) is unable to bind a hemimethylated DNA probe containing 3 GATC sites. The hemimethylated DNA probes containing 6 or 4 GATC sites differed in GATC site spacing and nucleotide sequences as compared with 3 GATC site, hemimethylated probes (7, 18). These differences might allow SeqA(E9K) to bind the 4 or 6 GATC sites, hemimethylated probe, rather than the 3 GATC site probe. It is also possible that defect in the interaction between SeqA(E9K) tetramers (Fig. 4B and Fig. 7) might cause SeqA(E9K) to require more GATC sites to form a stable complex than wild-type SeqA. The reduced number of shifted bands and its altered migration pattern by SeqA(E9K) as compared with wild-type SeqA suggest that interaction between SeqA(E9K) tetramers bound to GATC sites is different from the interaction between wild-type tetramers. This altered interaction between SeqA(E9K) tetramers may cause the formation of inactive (or less active) SeqA(E9K) aggregates.

Many proteins, including ClpB (26), beta -amyloid (27), human erythrocyte spectrin (28), and cowpea chlorotic mottle viral (CCMV) capsid protein (29), exert their biological functions through oligomerization or multimerization in a concentration-dependent manner. The reversible aggregation of SeqA tetramers may contribute to the formation of SeqA foci necessary for the segregation of chromosomal DNA. Aggregation may also be important in binding of SeqA to hemimethylated DNA. Although many GATC sequences are mostly clustered within and near oriC, the SeqA foci is not dependent upon oriC (10). However, hemimethylation of GATC sites is a requirement for proper SeqA foci formation. If increase in SeqA concentrations is insufficient during cell cycle for foci formation, these observations suggest that a factor(s) may trigger or aid the in vivo SeqA aggregation for foci formation on the hemimethylated chromosome.

    ACKNOWLEDGEMENT

We thank Dr. D. Ladant for DHP1, pT18-zip, and pT25-zip and Gillian Newman for careful editing of this manuscript.

    FOOTNOTES

* This work was supported in part by Grant 1999-1-209-004-5 from the Basic Research Program of Korea Science and Engineering Foundation and by a grant from Life Phenomena and Function Research of Korea Institute Science and Technology Evaluation and Planning.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by BK21 Research Fellowship from the Korean Ministry of Education.

** To whom all correspondence should be addressed. Tel.: 82-2-880-7524; Fax: 82-2-874-1206; E-mail: dshwang@plaza.snu.ac.kr.

Published, JBC Papers in Press, July 16, 2001, DOI 10.1074/jbc.M101339200

2 J. S. Han and D. S. Hwang, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DTT, dithiothreitol; DSS, dissucinimidyl suberate; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Messer, W., and Weigel, C. (1996) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., ed), 2nd Ed. , pp. 1580-1582, American Society for Microbiology, Washington, D. C.
2. Kornberg, A., and Baker, T. A. (1992) DNA Replication , 2nd Ed. , pp. 521-524, W. H. Freeman and Co., New York
3. Geier, G. E., and Modrich, P. (1979) J. Biol. Chem. 254, 1408-1413
4. Campbell, J. L., and Kleckner, N. (1990) Cell 62, 967-979
5. Lu, M., Campbell, J. L., Boye, E., and Kleckner, N. (1994) Cell 77, 413-426
6. Slater, S., Wold, S., Lu, M., Boye, E., Skarstad, K., and Kleckner, N. (1995) Cell 82, 927-936
7. Brendler, T., and Austin, S. (1999) EMBO J. 18, 2304-2310
8. Brendler, T., Sawitzke, J., Sergueev, K., and Austin, S. (2000) EMBO J. 19, 6249-6258
9. Boye, E., Stokke, T., Kleckner, N., and Skastad, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12206-12211
10. Hiraga, S., Ichinose, C., Niki, H., and Yamazoe, M. (1998) Mol. Cell 1, 381-387
11. Onogi, T., Niki, H., Yamazoe, M., and Hiraga, S. (1999) Mol. Microbiol. 31, 1775-1782
12. Skarstad, K., Lueder, G., Lurz, R., Speck, C., and Messer, W. (2000) Mol. Microbiol. 36, 1319-1326
13. Karimova, G., Pidoux, J., Ullman, A., and Ladant, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5752-5756
14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
15. Boye, E., and Löbner-Olesen, A. (1990) Cell 62, 981-989
16. Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130
17. Hwang, D. S., Thöny, B., and Kornberg, A. (1992) J. Biol. Chem. 267, 2209-2213
18. Kang, S., Lee, H., Han, J. S., and Hwang, D. S. (1999) J. Biol. Chem. 274, 11463-11468
19. Lee, H., Kim, H. K., Kang, S., Hong, C. B., Yim, J., and Hwang, D. S. (2001) Mol. Gen. Genet. 264, 931-935
20. Callaghan, J., Simonsen, A., Gaullier, J. M., Toh, B. H., and Stenmark, H. (1999) Biochem. J. 338, 539-543
21. Cadwell, R. C., and Joyce, G. F. (1995) in PCR Primer: A Laboratory Manual (Dieffenbach, C. W. , and Dveksler, G. S., eds) , pp. 581-589, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
22. Miller, J. H. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
23. Sonnenburg, W. K., Seger, D., Kwak, K. S., Huang, J., Charbonneau, H., and Beavo, J. A. (1995) J. Biol. Chem. 270, 30989-31000
24. Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, Part II , pp. 560-562, W. H. Freeman and Co., San Francisco
25. Marians, K. J. (1996) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., ed), 2nd Ed. , p. 753, American Society for Microbiology, Washington, D. C.
26. Barnett, M. E., Zolkiewska, A., and Zolkiewski, M. (2000) J. Biol. Chem. 275, 37565-37571
27. Sian, A. K., Frears, E. R., El-Agnaf, O. M., Patel, B. P., Manca, M. F., Siligardi, G., Hussain, R., and Austen, B. M. (2000) Biochem. J. 349, 299-308
28. Morrow, J. S., and Marchesi, V. T. (1981) J. Cell Biol. 88, 463-468
29. Zlotnick, A., Aldrich, R., Johnson, J. M., Ceres, P., and Young, M. J. (2000) Virology 277, 450-456
30. Weitao, T., Nordström, K., and Dasgupta, S. (1999) Mol. Microbiol. 34, 157-168
31. Weitao, T., Nordström, K., and Dasgupta, S. (2000) EMBO Rep. 1, 494-499

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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