Purification of a DNA Replication Terminus ( t e r ) Site-binding Protein in Escherichia coli and Identification of the Structural Gene*

In Escherichia coli cells, there is a protein that spe- cifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks pro- gress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plas-mid-free cells. With improvement of the promoter re- gion of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2000-fold. Using these overproducing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter pro- tein revealed a specific binding to the ter sequence. A DNA replication terminus (ter) site on replicons is the position at progress of the DNA replication fork is either arrested or is severely impeded. The ter sites required for termination of DNA replication are present the plasmid Escherichia and Bacillus subtilis

A DNA replication terminus (ter) site on replicons is the position at which progress of the DNA replication fork is either arrested or is severely impeded. The ter sites required for termination of DNA replication are present on the plasmid R6K genome and also on the bacterial chromosome of Escherichia coli and Bacillus subtilis (Lovett et al., 1975;Crosa et al., 1976;Weiss and Wake, 1983;Iismaa et al., 1984;Monteiro et al., 1984;Kuempel et al., 1977;Louarn et al., 1979). Similar sites have been found in yeast and plant cells (Brewer and Fangman, 1988;Hernandez et al., 1988). Thus, the ter site may play an important physiological role(s).
To block the progress of the DNA replication fork, two factors are required, one of which is the ter sequence on the DNA molecule. Properties of the ter site present in the E. coli system are as follows. (i) All ter sequences are essentially the same, and their consensus 22-bp' sequence is 5'-(A/T)(G/

T)TAGTTACAACAPy(A/T/C)C(A/T)(A/T)(A/T)(A/T)(A/
T)(A/T)-3'. The sequence was represented by ( ) (Horiuchi Hidaka et al., 1988;Hill et al., 1988b). (ii) The sequence has activity that inhibits travel of the replication fork in a specific direction Hidaka et al., 1988;Hill et ai., 1988b). In the above orientation, only the fork, traveling from right but not left, is inhibited. (iii) A pair of the two ter sites is arranged in an inverted position. In the R6K plasmid, a pair of two terR sites (terR1 and terR2) is arranged in an inverted position ( "++) 73 bp apart Hill et al., 1988b); in the E. coli chromosome, four terC sites (terC1,2,3, and 4 ) are located at the opposite region of the unique replication origin (oriC) and are arranged as ("++I-) 275 kilobases apart between the nearest pair of the terC sites . (iv) When the terC site is cloned into the ColEl derivative vector in the orientation in which the unidirectional replication fork starting from vector's origin is blocked at the terC site, presence of the site reduces the copy number of the hybrid plasmid because the site prevents the plasmid from completing DNA replication .
The ter-binding protein (Ter protein) is another factor essential for termination reaction. The ter-binding activity of the protein is controlled by a gene that we named tau (Kobayashi et al., 1989) or tus by Hill et al. (1988aHill et al. ( , 1989; in cells carrying the plasmid on which the tau gene was located, the ter-binding activity was enhanced about 8-fold compared with the plasmid-free cells. On the other hand, in the tau-defective cells, which showed the termination-less phenotype, no terbinding activity was evident, thereby suggesting that tau might be the structural gene for the Ter protein. Hill et al. (1989) determined the nucleotide sequence of the tus gene, the expected molecular mass of the product of which was about 36 kDa. Complementarily, Sista et al. (1989) purified ter-binding protein, the molecular mass of which was somewhat less than 40 kDa. From footprint experiments, they determined the sequence of the ter site covered by the protein.
Although these data suggested that tau (tus) might be the structural gene for the Ter protein, conclusive evidence was not obtained.
We report here the Ter protein-overproducing system, purification of the Ter protein, and identification of the structural gene (tau) for the protein. We confirmed in DNase I footprinting experiments that the Ter protein does specifically bind to the ter sequence.

MATERIALS AND METHODS
Procedures-Restriction endonucleases, T4 ligase, T4 polynucleotide kinase, and DNA polymerase large fragment (Klenow) were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). DNA ligation was done according to procedures outlined by the manufacturer. The composition of media used was as described (Miller, 1972). Synthesis of oligonucleotides was as described .
DNA Preparation-Plasmid and M13 replicative form DNA were isolated by the alkaline lysis procedure of Ish-Horowicz and Burke (1981). Some plasmid DNA were purified by equilibrium banding in CsCl gradients in the presence of ethidium bromide. Cells were transformed by the method of Kushner (1978). DNA fragments used for gel retardation assay were purified electrophoretically on acrylamide gels. The appropriate fragment was cut from the gel with ethidium bromide and obtained by the method using DEAE-paper from the gel (Dretzen et al., 1981). Single-stranded M13 templates were prepared by the procedure of Nakamaye and Eckstein (1986).
Gel Retardation Assay-Procedures used were essentially the same as described by Wang et al. (1987) and by our group (Kobayashi et al., 1989). As the DNA substrate for the assay, we used the 141-base pair AluI-HaeIII fragment, on which was located one of a pair of two terminus sites, terRl. The fragment was end-labeled with T4 polynucleotide kinase according to in Maniatis et al. (1982). Construction of Ter Protein-overproducing Plasmid-pUC9-5.0(-) was the parental plasmid used . The plasmid is a pUC9 derivative in the EcoRI site of which the 5.0-kilobase EcoRI fragment carrying terC2 site and the tau gene was inserted. A 2.7kilobase HindIII-EcoRI subfragment of the 5.0-kilobase fragment was recloned into M13mplO replicative form DNA, and the recombinant phage was used for the oligonucleotide-directed in vitro mutagenesis system (version), supplied by Amersham International plc. The mutagenesis procedures are based on the method of Eckstein and coworkers (Sayers et al., 1988). For first and second mutagenesis, the oligonucleotides 5'-AAATAAGTATGTT@TAACTAAA-3'(22 mer) and 5'-TAACTAAAC@GGTTAATATT-3' (20-mer) were used, respectively. The nucleotide circled indicates each mutation point. Identification of the mutant progeny was screened in hybridization experiments with the mutant oligonucleotide, the procedures of which were according to the method given by the supplier. Introduction of the first mutation was confirmed by DNA sequencing of the target site and surrounding areas. The replicative form DNA of the two mutant progeny phages was prepared, digested with Hind111 and EcoRI, and recloned into the corresponding site of the pUC9 vector.
The resulting two plasmids carrying single and double mutations (pKHG300) were used as the Ter protein overproducers.
Overproduction and Purification of Ter Protein-The Ter protein overproducer plasmid, pKHG300, was introduced into the JM83 host strain, and an ampicillin-resistant transformant was isolated. The method used for cell lysate preparation was essentially the same as described in Wickner et al. (1974). A fresh colony of the strain was inoculated into 50 ml of LB-ampicillin broth and shaken slowly at 30 "C to reach an optical density of about 0.7 at 660 nm. The cells were collected at low speed centrifugation at 5,000 rpm for 10 min, resuspended in 0.1 ml of Buffer B (50 mM Tris-HC1 (pH 7.51, 10% sucrose (total = 380 pl)), placed in liquid nitrogen, and kept in a -80 "C deep freezer. The cells were thawed in an ice bath to which 12 pl of Buffer B containing 4 mg/ml lysozyme and then 6 pl of 5 M NaCl were added. The mixture was left to stand for 30 min in the ice bath, for 90 s at 37 "C, and then centrifuged at 4 "C at 15,000 rpm. The supernatant (fraction I, 150 pl) was transferred to a different microcentrifuge tube, the volume adjusted to 500 pl with Buffer B, and ammonium sulfate powder was added to reach a concentration that would achieve a 50% saturation. After standing in an ice bath for 1 h, the centrifuged and precipitated fractions were dissolved in 500 pl of Buffer A (50 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol) and dialyzed overnight at 4 "C with Buffer A containing 20 mM NaCl (fraction 11, 530 PI). Fraction 11 (500 p l ) was applied to a DEAE-Sephacel (Pharmacia LKB Biotechnology Inc.) column (1 ml), and the flow-through fraction at 0.1 M NaCl (fraction 111, 3 ml) was directly applied to a heparin-Sepharose CL-GB column (1 ml) (Pharmacia). The column was then washed with 4.5 column volumes of Buffer A containing 0.3 M NaCl and eluted sequentially with Buffer A containing 0.4, 0.5, 0.6, and 1.0 NaCI. Ter protein was eluted at 0.5-0.6 M NaCl (fraction IV, 3 ml).
Protein Concentration Determination-Protein concentration was determined using a BCA protein assay (Pierce) according to the method of Smith et al. (1985). Bovine serum albumin was used as the standard. Standards were prepared with the same concentration of enzyme buffer ingredients present in the sample.
DNA Sequence Analysis-DNA sequences were carried out on a single-stranded M13 using the dideoxy chain termination method (Sanger et al., 1977). DNA sequence kits were obtained from United States Biochemical Corp. Appropriate 20-mer synthetic oligonucleotides were synthesized as additional primers for further sequencing along the same template.
DNase I Footprinting Assay-The assay was performed according to the method of Galas and Schmitz (1978). The reaction mixture (100 pl) contained 10 pl of 10 X reaction buffer (100 mM Tris-HC1 (pH 7.5), 500 mM NaCI, 10 mM dithiothreitol, 5 mM EDTA, 25 mM MgCIZ, 50% glycerol); 2 p l of 32P end-labeled A h 1 (216-base pair) DNA fragment (-5.3 ng; 38 fmol); 10 pl (0-32 pmol) of Ter protein solution in dilution buffer (10 mM Tris-HC1 (pH 7.5), 50 mM NaCI, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol); 78 pl of distilled water was incubated a t 25 "C for 30 min. Two pl of lo-' diluted solution (5 mM CaClz, 10 mM MgCl,) of DNase I (1 mg/ml in 0.15 M NaC1, 50% glycerol, 1950 units/ml; Sigma type 11) was then added, a partial digestion of the DNA fragment was carried out at 25 "C for 5 min, and the action was terminated by adding 25 pl of stop solution (1.5 M sodium acetate (pH 5.2), 20 mM EDTA, 100 pg/ml sonicated calf thymus DNA). One hundred pl of phenol saturated with 100 mM Tris-HC1 (pH 8.0) was added, Vortex mixed for 30 s, then centrifuged for 5 min at 15,000 rpm. To the aqueous phase thus obtained we added 300 p1 of ethanol, left the preparation to stand at -70 "C for 30 min, and the pellet was collected by centrifugation for 30 min at 15,000 rpm at 4 "C and then dissolved with 100 p1 of 0.3 M sodium acetate. After adding 250 p1 of ethanol, the precipitation was repeated, the pellet was washed once with 200 p1 of 70% ethanol and dried. Four pl of loading buffer (80% (v/v) deionized formamide, 50 mM Tris-HC1 (pH 8.3), 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromphenol blue, 1 mM EDTA) was added, 2 pl of the sample was applied to a polyacrylamide sequence gel and electrophoresed. As control samples, the same DNA fragment was used for base-specific cleavage reactions, as described by Maxam and Gilbert (19801, and electrophoresed. The gel was then dried and exposed to x-ray film (Kodak, X-Omat).
Amino Acid and Amino-terminal Sequence Analysis-Samples were hydrolyzed in tubes sealed under reduced pressure with 5.7 M HCI for 24,48, and 72 h at 110 "C and with 3 M mercaptoethanesulfonic acid for 24 h at 110 "C. After evaporation, the hydrolysates were analyzed on a Hitachi model L8500 high speed amino acid analyzer (Spackman et al., 1958). Half-cystine was determined as cysteic acid after performic acid oxidation. Amino acid sequences were determined using an Applied Biosystems 47719 gas-phase sequenator.

RESULTS
Nucleotide Sequence of the tau Gene-We had already identified the tau gene, defective mutants of which showed termination-less phenotype (Kobayashi et ai., 1989). This gene was located on a 2.7-kilobase HindIII-EcoRI fragment situated at 35.5 min of the standard E. coli map (Bachmann, 1972;Kohara et al., 1987). Within or near the end of the tau gene was the terC2 site, one of the four DNA replication terminus (terC) sites on E. coli genome Hill et ai., 198813). The tau gene was localized within the 0.75-1.0-kilobase region from the terC2 site by insertional mutagenesis. The 2.7-kilobase HindIII-EcoRI fragment and its subfragment were cloned into the M13 family of vectors. Subsequent dideoxy sequencing of these clones revealed a single unique open reading frame in the region predicted to contain the tau gene, as shown in Fig. 1. In the promoter region of the gene, -35, -10, and ribosome-binding (Shine-Dalgarno) sequences were also present (Rosenberg and Court, 1979). Interestingly, the -10 region and ribosome-binding site overlapped with the terC2 sequence. The molecular weight of the open reading frame product was about 35,700. The sequence of the gene is exactly the same as that of the tus gene sites for EcoRI, HindIII, PuuII, and BglI. a, restriction map shows only relevant Ready-made oligonucleotides or synthetic oligonucleotides with the sequence at start site of the arrows shown were used as was the dideoxynucleotide chainterminator method (Sanger et al., 1977). b, the underlined amino acids (without m e t ) represent the amino-terminal sequence determined for the purified 36-kDa protein, -35, -10, and ribosomalbinding (Shine-Dalgarno) sites are underlined. The terC2 sequence is represented by bold letters. Two mutation changes, which convert the original tau plasmid to the Ter-overproducing plasmid, are shown.   reported by Hill et al. (1989) although there are differences in the region downstream of the tau gene.

C G A T T C G G T T C A A T A C C C A C T G C G C A G T G T T T G T T R A R A C T
Overproduction of the Ter Protein-We observed that crude extracts of cells with the plasmid-carrying tau gene had about an 8-fold higher ter-binding activity than that seen in the plasmid-free cells (Kobayashi et al., 1989). To overproduce the protein, we introduced two mutations in vitro at the promoter site of the tau gene on the plasmid, as shown in Fig.  2. The first mutation was introduced within the terC2 sequence. A low copy number of the pUC9-terC2 plasmid (a common property found in all Ter-active pUC-terC plasmids) reached a normal level (data not shown). The second mutation was made within the ribosome-binding sequence. Resulting plasmids were introduced into the JM83 host, and binding activity of the crude extract was measured. A plasmid with single and double mutations enables host cells to overproduce 500 and over 2000-fold the ter-binding activity of the plasmidfree control strain, respectively (data not shown). These results suggest that the terC2 area might be an operator site, at which Ter protein binds tightly and inhibits the expression of the tau gene as well as progress in the DNA replication fork; that is to say, the tau gene may be autoregulated. Using the overproducer strain as the enzyme source, we purified the Ter protein.
Purification of Ter Protein-In the overproducing cells, a unique protein with a molecular mass of 36 kDa was overproduced (Fig. 3). Densitometric scanning of the gel led to an estimation of about 14% of the total protein. The corresponding protein band was not present in the sample of cells carrying only the vector (data not shown). This estimated molecular mass is close to the 40 kDa already reported (Sista et al., 1989). We attempted to purify the Ter protein by   following the 36-kDa protein band through SDS-polyacrylamide gel electrophoresis analysis. Purification of the protein was attained by the procedures shown in Table I. The 36-kDa protein behaved as a basic protein; it did not bind to DEAE-Sephacel but did bind tightly to heparin-Sepharose, even in . Co-purification of ter-binding activity with the 36-kDa Ter protein through gel filtration. Sample (200 pl) of fraction IV was dialyzed with buffer (50 mM Tris-HCI (pH 9.0), 1 mM dithiothreitol, 1 mM EDTA, 5% (v/v) glycerol, 300 mM NaCI) overnight, applied to Superose 12HR 10/30 (Pharmacia), and eluted with the same buffer. Absorbance a t 280 nm was measured with a UV detector, and a sample of the fraction (0.5 ml) was analyzed by SDS-polyacrylamide gel electrophoresis and gel retardation assay to measure the ter-binding activity. a, absorbance a t 280 nm; b, SDSpolyacrylamide gel electrophoresis analysis; c, ter-binding activity. DNA substrate and DNA-protein complex indicate the positions corresponding to the substrate DNA fragment (141 base pairs) and DNA-protein complex, respectively. the presence of 0.1 M NaCl. Here we defined 1 unit of the terbinding activity as that which could shift half of the terR1containing DNA substrate (9.6 fmol) added to the band position corresponding to the DNA-protein complex, through the gel retardation assay, as shown in Fig. 4c. Using this enzyme unit, we determined the specific activity of the protein fraction obtained a t each stage of purification. Table I shows that the specific activity of ter-binding protein in the initial step was raised to 5-fold at the final step. This is near the expected level (7.14-fold). The 36-kDa protein in fraction IV, eluted from the heparin-Sepharose with 0.5-0.6 M NaCl, was further purified through gel filtration, and the ter-binding activities in each fraction were measured. As shown in Fig. 4, three different parameters (absorbance a t 280 nm, a protein band of 36-kDa, and ter-binding activity) completely matched. We concluded that the 36-kDa protein is a Ter protein.
The amino-terminal amino acid sequence was determined for the purified 36-kDa protein in step IV using automated Edman degradation. Amino-terminal 45 residues, albeit with the interruption of 3 unidentified amino acids (including m e t ) , is in perfect agreement with that deduced from the DNA sequence of the tau gene (Fig. 1). The amino acid composition (Table 11) as well as the total molecular mass further supported the conclusion that the open reading frame, that is tau gene, encodes the Ter protein with a molecular mass of 36 kDa.
DNA-binding Site of the Ter Protein-To determine the binding site of the Ter protein, a DNase I footprinting experiment using purified Ter protein was done. Terminus sites (terRI and terR2) of the plasmid R6K, essential for termination reaction, were located on 216 bp of AluI fragment Horiuchi and Hidaka, 1988). One end, closer to the terR2 site of the fragment, was labeled with '"P, mixed with the Ter protein, and partial DNase I digestion followed by polyacrylamide gel electrophoresis was performed. Fig. 5 shows that the Ter protein binds to the two sites that correspond exactly to terR1 and terR2. Since the sequences are highly homologous and placed in an inverted arrangement, two regions covered by the protein can be regarded as regions  of both DNA strands of either of the terR sites covered by the protein as shown in Fig. 6. The terR site was first identified as the 2L"bp sequence required for the termination reaction in vivo and to which Ter protein binds.

DISCUSSION
We obtained convincing evidence that the tau gene encodes the ter-binding protein (Ter protein). This Ter protein recognized and bound to all ter sites (at least two terR and four terC sites), and the resulting Ter protein-ter sequence complex can block the DNA replication fork on the DNA molecule.
The sequence analysis of the tau gene suggested that there might be a site (terC2) at the promoter region of its own gene, to which Ter protein binds tightly. Since Ter protein overproduction was attained by a mutational destruction of the termination activity of the terC2 sequence, it is most probable that Ter protein might be a repressor for expression of its own gene; that is to say the tau gene is under autoregulatory control. The terC2 and -10 sequence apparently overlap, as shown in Fig. 1, and the overlapped sequence is covered by the Ter protein (Fig. 6). Thus, the binding of Ter protein to the terC2 site prevents RNA polymerase from interacting with the promoter site. This possibility was suggested by Hill et al. (1989). This autoregulatory control might maintain constant the quantity of the Ter protein. If such is indeed the case, then the Ter protein is exceptional; the protein plays two roles at the same site, terC2, one is a replication blocker, and the other is the repressor for its own gene expression.
However, with regard to Ter protein overproduction by the mutant plasmid, another explanation would have to be considered. As shown in Fig. 2, orientation of the replication fork starting from the pUC vector replication origin is that blocked by the terC2 site; the copy number of the parental plasmid is low. Mutational inactivation of the terC2 sequence resulted in reversion of its low copy number to normal level.' Furthermore, orientation of the tau gene transcription is the same as that of lac2 gene on the vector plasmid. Thus, a high dosage of the tau gene and its high expression under lacZ control would make the gene product overproduce on the mutant plasmid. Evidence nonsupportive of this idea is that while the overproducing plasmid was introduced into JM109 (ladq: lactose repressor-overproducing gene), the high level expression of the Ter protein as in the JM83 host was maintained: thereby suggesting that tau gene expression is out of lac2 control. Another reason is that the nonmutant tau gene on the pUC plasmid and with normal copy number was able to overproduce its product only &fold (Kobayashi et al., 1989). Thus, autoregulation seems to be a more tenable explanation. Hill et al. (1989) reported that the primary promoter of the tus (tau) gene is located at least 1200 base pairs upstream of the tus gene and that the weak promoter was identified immediately upstream of the tus gene. However, their results did not exclude the existence of the autoregulatory circuit.
In B. subtilis, a similar ter system seems to be operative. Wake and co-workers found that the terC site of B. subtilis, a t which at least a clockwise replication fork was blocked, was located at the region opposite that of the origin of replication (Weiss andWake, 1983,1984). Sequence analysis of the region revealed that at the terC site, there is a long inverted repeat sequence homologous to the E. coli terC sequence (Carrigan et al., 1987). Adjacent to the terC site, there is an open reading frame capable of coding a basic protein with 122 amino acids, the defective mutant of which showed a termination-less phenotype (Iismaa and Wake, 1987;Smith and Wake, 1988; S. Takenaka and T. Horiuchi, unpublished data. '' M. Hidaka, unpublished data. Lewis and Wake, 1989). Recently this protein, like the E. coli tau product, was found to have terC-binding activity (Lewis et al., 1989), although they apparently share no homologous region (Hill et al., 1989).
Sista et al. (1989) purified the terR-binding protein of E.
coli and determined the binding site of the purified protein.
This protein may be the same as the one purified in our present study; however, the region covered by their protein is narrower than that shown in our Fig. 6, although both patterns are essentially the same. The different techniques used may account for the discrepancies; we used DNase I and they used copper-phenanthroline (Kuwabara and Sigman, 1987) for the footprinting experiment.
The molecular structure of the Ter protein-ter sequence complex seems to be unique since the Ter protein is the first example of a DNA-binding protein that can block movement of the DNA replication fork. Particulars regarding this block and the molecular mechanisms involved in progress of the replication fork at the replication point are now being investigated.