Functional Domains of Rts1 and P1 RepA Proteins for Initiation of Replication

PLASMID

40, 140 –149 (1998) PL981360

ARTICLE NO.

Functional Domains of Rts1 and P1 RepA Proteins for Initiation of Replication Yong Fang Li, Tetsuya Hayashi, and Yoshiro Terawaki1 Department of Bacteriology, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, 390, Japan Received February 17, 1998; revised May 1, 1998 Rts1 RepA and P1 RepA are trans-acting proteins essential for initiation of replication of Rts1 and P1 plasmids, respectively. We recently found that P1 RepA bound in vitro to the Rts1 replication origin as strongly as Rts1 RepA and activated the origin in vivo. However, the ori activation was quite inefficient. This study shows that by replacing a small region of P1 RepA with the corresponding region of Rts1 RepA, the efficiency of Rts1 ori activation increased markedly. Interestingly, the same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin. Thus, a region essential for efficient activation of the replication origin was assigned to the P1 RepA molecule as well as to the Rts1 RepA molecule. The region was distinct from a domain necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the respective origin. © 1998 Academic Press

Mini-Rts1, a minimal replicon of the plasmid Rts1, consists of three important components (Kamio et al., 1984). They are (1) the replication origin ori, (2) a gene, repA, and (3) the incI iterons. The repA gene encodes the initiator protein RepA which binds to the direct repeated sequences (iterons) in ori, leading to initiation of Rts1 replication. In the ori region, tandem DnaA boxes and four GATC repeats are located upstream of the iterons. The incI iterons, located downstream of repA, are involved in negative control of the plasmid replication. The replicon structure is quite similar to that of mini-P1, the minimal replication region of the phage P1, which contains ori iterons with DnaA boxes and GATC repeats, a repA gene, and incA iterons (Abeles et al., 1984). In addition, the amino acid sequence of Rts1 RepA, consisting of 288 amino acids, shares 60% homology with that of P1 RepA consisting of 286 amino acids. Both RepA proteins inhibit specifically the replication of their respective plasmid when an excess amount of the protein is supplied in trans (Muraiso et al., 1990; Terawaki et al., 1990). Thus, RepA proteins of Rts1 and P1 exhibit dual functions in the replication machinery. They induce 1

To whom correspondence should be addressed. Fax: (81) 263–37–2616. E-mail: [email protected]. 0147-619X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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replication positively by binding to the ori sequence and regulate repA negatively by binding to the repA promoter region (Kamio et al., 1988; Sozhamannan and Chattoraj, 1993). For negative regulation of replication, pairing or “handcuffing” of RepA–DNA (ori and/or inc iterons) complexes might be involved, as proposed for R6K and RK2 as well as P1 (Miron et al., 1994; Blasina et al., 1996; Chattoraj et al., 1988; Pal and Chattoraj, 1988; Abeles and Austin, 1991; Mukhopadhyay et al., 1994). To investigate the functional domains of Rts1 RepA, we recently constructed hybrid proteins of Rts1 RepA with P1 RepA such that the N-terminal region was from Rts1 RepA and the C-terminal region was from P1 RepA (R/P type hybrid). From this study we have tentatively determined two subregions in Rts1 RepA related to initiation. One, near the N-terminal region, between amino acid residues 113 and 129, is important for the origin binding in vitro, and another, between residues 177 and 206, is required along with the DNA binding domain for origin activation in vivo (Tabuchi et al., 1995). In addition, we recently found that P1 RepA bound in vitro to Rts1 ori as strongly as Rts1 RepA, and could induce in vivo replication from the Rts1 origin, although the ori activation

DOMAINS OF Rts1 AND P1 RepA

was inefficient (Li et al., 1997). In contrast, Rts1 RepA showed neither in vitro binding to P1 ori nor in vivo activation of the origin. To gain more insight into the structure and initiation function of the RepA molecule, in this study, we replaced a central portion of P1 RepA with the corresponding portion of Rts1 RepA (P/R/P type hybrid). P/R-type hybrid proteins consisting of the N-terminal portion from P1 RepA and the C-terminal portion from Rts1 RepA (P/R type hybrid) were also constructed. The hybrid proteins obtained were examined for in vitro binding to Rts1 and P1 origins as well as in vivo activation of the origins. MATERIALS AND METHODS Bacterial Strains and Phages Escherichia coli strain HB101 (F2 leuB6 supE44 thi-1 hsdS20 recA13 ara-14 proAB lacY1 galK2 rpsL20 xyl-5 mtl-1) (Boyer and RoullandDussoix, 1969) was used for plasmid DNA construction. Strain JG112 (polA lacY thy str) (Miller et al., 1978) was used for the Rts1 ori and P1 ori activation assay, and JM109 [recA1 supE22 endA1 hsdR17 gyrA96 relA1 thi D(lac-proAB) F9 (traD36 proAB lacIq lacZDM15)] (Yanisch-Perron et al., 1985) was the host for M13 phage. M13mp18 phages (Yanisch-Perron et al., 1985) were used as cloning vectors for PCR2 fragments. Media and Chemicals Luria–Bertani medium (LB broth, Difco Laboratories, Detroit, MI) was used for transformation, Penassay broth (Difco) for selection of transformants, and 2YT medium (Miller, 1972) for isolation of plasmid DNA. The following antibiotics were included in the medium when needed: ampicillin (Ap, 30 mg/ml), spectinomycin (Sp, 30 mg/ml). T4 DNA ligase was obtained from Takara Shuzo, Kyoto, Japan. Restriction enzymes were from Takara Shuzo, New England BioLabs, Inc. (Beverly, MA) or Bethesda Research Laboratories, Inc. (Gaithersburg, MD). All the enzymes were used as rec2 Abbreviations used: PCR, polymerase chain reaction; LB broth, Luria–Bertani medium; Ap, ampicillin; Sp, spectinomycin.

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ommended by the suppliers. [g-32P]ATP, used for end labeling DNA fragments, was purchased from Amersham (Buckinghamshire, England). DNA Isolation and Gene Manipulation Plasmid DNA was isolated by the method of Humphreys et al. (1975). Transformation was carried out essentially by the method of Dower et al. (1988). Briefly, recipient cells were grown at 37°C in SOB medium (2% Bacto-tryptone, 0.5% Bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl) to a cell density of 0.7– 0.8 at OD550. The cells were washed extensively and finally resuspended in cold 10% glycerol. Plasmid DNA was introduced into the cells by electroporation (12.5 kV/cm) with a Cellject basic electroporation system (Equi Bio). The electroporated cells were grown in SOC medium (SOB with 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) at 37°C for 1 h and seeded onto the selective plates. Restriction endonuclease fragments were purified from agarose gels with a Prep-A-Gene DNA purification kit (Bio-Rad Laboratories, Richmond, CA). Polymerase Chain Reaction The PCR was carried out with a DNA Thermal Cycler (Model IJ1000; Takara Shuzo) and the GeneAmp PCR reagent kit with Ampli-Taq DNA polymerase (Perkin–Elmer Cetus, Norwalk, CT). Various oligonucleotides used as the primer were synthesized by the phosphoramidite method as described previously (Zeng et al., 1990), or obtained from Bio-Synthesis Inc., Lewisville, Texas. DNA Sequence Analysis The nucleotide sequences of PCR products inserted in M13 phages and the repA regions in the pGB:LXn plasmids were determined by the dideoxy chain termination method (Sanger et al., 1977) with a DNA sequencer (Model 370A, Perkin–Elmer). DNA sequencing reagents and enzymes were obtained as a kit from Perkin– Elmer.

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LI, HAYASHI, AND TERAWAKI TABLE 1 Plasmids Used in This Study

Plasmid

Description

Reference or source

pBR322 pGB2 pTW22 pTW22:Rts1ori pBR322:P1ori pALA619 pTLXnd pBR322:LX01 pBR322:LXn pGB:LX01

Apr, Tcr pSC101 derivative; Spr pBR322 1 rrnB transcriptional terminator; Apr pBR322 1 Rts1 ori (1441–1194)a; Apr pBR322 1 P1 ori (290–608)b; Apr pGB2 1 promoter of pBR322 (4168–29)c 1 P1 repA; Apr pTW22 1 P1/Rts1 or P1/Rts1/P1 hybrid repA; Apr pBR322 1 Rts1 repA (1158–260)a without incI region; Apr pBR322 1 P1/Rts1 or P1/Rts1/P1 hybrid repA; Apr pGB2 1 promoter of pBR322 (4168–29)c 1 Rts1 repA (1158–260)a without incI region; Spr pGB2 1 promoter of pBR322 (4168–29)c 1 P1/Rts1 or P1/Rts1/P1 hybrid repA; Spr

Bolivar et al., 1977 Churchward et al., 1984 Tabuchi et al., 1995 Tabuchi et al., 1995 Li et al., 1997 Abeles et al., 1990 This study This study This study This study

pGB:LXn

This study

a

Numbers in parentheses are mini-Rts1 coordinates (Kamio et al., 1984). Numbers in parentheses are mini-P1 coordinates (Abeles et al., 1984). c Numbers in parentheses are pBR322 coordinates (Bolivar et al., 1977). d Number (n 5 15, 151, 152, 153, 17) indicates P1/Rts1 and P1/Rts1/P1 hybrid repA. b

Construction of Plasmids Plasmids used in this study are listed in Table 1. To construct plasmids carrying various hybrid repA, a series of fragments were synthesized by PCRs. The synthesized fragments had restriction endonuclease sites at both ends, which were used to construct each hybrid repA without insertion of any additional amino acids in the hybrid RepA (Fig. 1). The synthesized fragments were cloned into the polylinker sites of M13mp18, and their DNA sequences were confirmed by sequencing. A series of pTLXn plasmids carrying various hybrid repA were constructed as follows: pTLX15 was first constructed by replacing the HindIII– ClaI fragment of pTWX01 (Tabuchi et al., 1995) containing the promoter and N-terminal half of Rts1 RepA [mini-Rts1 coordinates: 1191–697 (Kamio et al., 1984)], with the synthesized HindIII–ClaI fragment carrying the N-terminal sequence of P1 repA [mini-P1 coordinates: 608– 1095 (Abeles et al., 1984)]. Note that the hybrid repA lacks its own promoter since the HindIII site of the P1 fragment is located just downstream of the P1 repA promoter sequence. To construct pTLX17 and pTLX 151, the relevant part of repA on pTLX15 was replaced with the EcoRI–EcoRV

fragment (Rts1: 790–361) for pTLX17 and the XbaI–BamHI fragment (P1: 1278–1527) for pTLX151. pTLX152 was constructed by substituting the EcoRI–BamHI region of pTLX15 with the synthesized EcoRI–SacI fragment of pTLX15 (P1: 1000–1095 plus Rts1: 697–605) and SacI– BamHI fragment (P1: 1193–1527). To construct pTLX153, the EcoRI–BamHI fragment of pTLX15 was substituted by two synthesized fragments, EcoRI–SacI (P1: 1000–1193) and SacI– BamHI (Rts1: 605–260), and then, the XbaI– BamHI part of this fragment was replaced with the synthesized XbaI–BamHI fragment (P1: 1278– 1527), finally resulting in pTLX153. To construct a series of pGB:LXn plasmids, each carrying the repA hybrids constructed above, the HindIII–BamHI fragment of the respective pTLXn plasmid was inserted into the HindIII–BamHI site of pBR322 (pBR322:LXn). pGB:LX01 carrying the wild-type Rts1 repA was constructed by inserting the synthesized HindIII–BamHI fragment (Rts1: 1158 –260) of pGB:X01 (Tabuchi et al., 1995) into the HindIII–BamHI site of pBR322 (pBR322:LX01). Subsequently, the SspI–BamHI fragment of each pBR322:LXn was cloned into the HincII– BamHI site of the pGB2 vector. Since the SspI–

DOMAINS OF Rts1 AND P1 RepA

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FIG. 1. Structure of pGB:LXn plasmids carrying hybrid repA genes. Bold arrows indicate the repA genes of P1 (open) and Rts1 (closed), encoding 286 amino acids (AA) and 288 AA, respectively. Hybrid proteins of P/R type RepA, 287 AA, consist of the N-terminal portion from P1 RepA and the C-terminal portion from Rts1 RepA; hybrid proteins of P/R/P type RepA, 286 AA, consist of the N-terminal and C-terminal portions from P1 RepA, but a midportion from Rts1 RepA. The restriction endonuclease during cutting sites at which recombination took place are shown. The numbers of amino acids from each RepA composing the hybrid proteins are indicated in parentheses (number from P1 RepA/number from Rts1 RepA; or number from P1 RepA/number from Rts1 RepA/number from P1 RepA).

HindIII fragment of pBR322 possesses a constitutive but uncharacterized promoter activity (Abeles et al., 1990) and the native promoter of repA was eliminated from all constructs during the plasmid construction, all the repA on the pGB:LXn plasmids are transcribed constitutively from the pBR-derived promoter. pALA619 carrying the wild-type P1 repA has the same structure as pGB:LXn plasmids (Abeles et al., 1990). Rts1 and P1 ori Activation Rts1 and P1 ori activation by hybrid RepA in trans was examined by transforming plasmid pTW22:Rts1ori or pBR322:P1ori into JG112 harboring hybrid RepA-producing plasmids pGB:LXn. The pBR322 origin present on these Rts1- and P1-ori plasmids is inactive in this polA host. These plasmids can replicate in this host only if the Rts1- and P1-RepA proteins are

functional. Ampicillin resistance, carried on the ori (Rts1 or P1) plasmid, was used to indicate Rts1 or P1 ori activating function of the hybrid proteins. Transformant colonies that developed on plates containing both Ap and Sp were examined for the stability of the ori plasmids. A single transformant colony was picked from the plate and resuspended into LB broth without drug. After 5 h of incubation at 37°C, the culture was streaked onto plates containing Sp and incubated overnight at 37°C. One hundred colonies from these plates were then examined for resistance to Ap. Preparation of Cell Extracts of Hybrid Proteins HB101 cells harboring the pGB:LXn plasmids were used for extraction of the hybrid proteins. The proteins were prepared by ammonium sulfate fractionation of cell lysates by the

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method for preparation of “fraction II” reported by Fuller et al. (1981) except that the dialysis buffer contained 50 mM Tris (pH 7.5), 1 M NaCl, 1 mM EDTA, 2 mM mercaptoethanol, and 10% (v/v) glycerol. The composition of the dialysis buffer was basically that described by Abeles (1986) for P1 RepA preparation. Protein concentrations were determined with the BioRad protein assay kit with bovine serum albumin as a standard. Immunoblot Analysis of Hybrid Proteins The concentrations of various “fraction II” proteins were adjusted to contain the same amount of total protein in each well and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis by the method of Laemmli (1970). Polypeptides in the gel were transferred to a nitrocellulose filter (pore size, 0.45 mm) by the method of Towbin et al. (1979). The filter was blocked with bovine serum albumin, treated with anti-Rts1 RepA or anti-P1 RepA antibody, and treated with alkaline phosphatase-conjugated anti-rabbit immunoglobulin G antibody (Promega Biotec, Madison, WI); color development was then done as recommended by the supplier. Electrophoresis Mobility Shift Assay for Hybrid Protein–DNA Binding The binding of hybrid proteins to DNA fragments was assayed as follows. The 10-ml binding mixture contained 20 mM Tris–acetate, pH 7.5, 15 mM magnesium acetate, 1 mM dithiothreitol, 0.5 mM ATP, 5 mM EDTA, 0.2 mg poly(dI– dC) (Pharmacia), 200 mM potassium glutamate, 5% (v/v) glycerol, 10 ng DnaJ protein, and 80 ng DnaK protein (Epicentre Technologies), 0.05 pmol 32P-end-labeled DNA fragment and various amounts of “fraction II” extracts containing hybrid proteins. The reactions were first assembled without the nucleic acids and incubated for 10 min at 20°C to activate the RepA protein. After addition of the DNA, the mixture was kept at 20°C for 20 min. Immediately after incubation, it was mixed with 5 ml dye buffer [10% Ficoll-400, 0.1% bromphenol blue, 10 mM Tris (pH 7.7), 1 mM

FIG. 2. Immunoblotting of wild-type and hybrid RepA proteins (fraction II extracts) with anti-Rts1 RepA antibody (A) and anti-P1 RepA antibody (B). Proteins were prepared from E. coli HB101 harboring the pGB:LXn plasmids. The same amount of (total) protein of fraction II was applied into the gel. Lane 1, Rts1 RepA; lane 2, P1 RepA; lane 3, RepALX15; lane 4, RepALX17; lane 5, RepALX151; lane 6, RepALX152; lane 7, RepALX153; lane 8, pGB2(vector).

EDTA], loaded onto a 5% polyacrylamide gel, and subjected to electrophoresis at 5 V/cm. The gel was dried and autoradiographed with Fuji Medical X-ray film at 270°C overnight. RESULTS Synthesis of Hybrid Proteins The fraction II extracts from cells harboring the repA hybrid plasmids were examined for the presence of proteins, corresponding in size to wild-type RepA, by immunoblotting with antiRepA (P1 and Rts1) antibodies. As shown in Fig. 2, a band was seen in the extracts containing any of the hybrid RepA. However, the concentration of each hybrid protein in the extract could not be estimated from this immunoblot, since the reactivity of each hybrid RepA to the RepA antibody was likely to be different from that of RepAwt. RepALX151 reacted weakly to both P1 RepA and Rts1 RepA antibodies. The weaker band does not necessarily mean a smaller amount of the protein, because the fraction II containing RepALX151 gave strong retarded bands in the electrophoresis mobility shift assay as will be shown below. The other P/R/P-type hybrid proteins, RepALX152 and

DOMAINS OF Rts1 AND P1 RepA

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FIG. 3. Binding of hybrid proteins to Rts1 ori. The 0.25-kb BglII–HindIII fragment, end-labeled with 32P (0.05 pmol DNA, 105 cpm), was mixed with the fraction II extracts containing the hybrid protein and allowed to bind for 20 min at 20°C as described under Materials and Methods. “mg of protein” indicates the concentration of total protein in fraction II.

RepALX153, reacted strongly to both RepA antibodies. This was expected, since P1 RepAwt reacted strongly not only to anti-P1 RepA antibody but also to anti-Rts1 RepA antibody. Binding in Vitro of Hybrid Proteins to Replication Origins The DNA fragments used for electrophoresis mobility shift assays were the 0.25-kb BglII– HindIII Rts1 ori fragment from pTW22:Rts1ori (Tabuchi et al., 1995), containing four 21-bp directly repeated sequences (iterons) along with four GATC repeats and the tandem DnaA boxes, and the 0.3-kb EcoRI–HindIII P1 ori fragment from pALA630 (Brendler et al., 1991), containing five 19-bp iterons with four GATC repeats and the tandem DnaA boxes. As shown in Fig. 3, all of the hybrid proteins except RepALX153 bound to the Rts1 ori almost as strongly as Rts1 RepAwt. Since all of the hybrid proteins were produced from the same promoter, we assume that their concentrations are about the same. Thus the reduced binding of RepALX153 would not be due to the presence of less protein. The two P/R-type hybrid proteins bound the P1 origin differently (Fig. 4): RepALX15 yielded clear though less intense

retarded bands, but RepALX17 showed no binding. The lack of binding by RepALX17 does not indicate its instability or improper folding, because it showed a strong binding to Rts1 ori, comparable to the binding by Rts1 RepAwt as shown in Fig. 3. Therefore, we can conclude that a region of P1 RepA that is involved in specific binding to P1 ori is between residues 113 and 144. In addition, the C-terminal region of P1 RepA appears to be required for efficient binding to the origin, since the three P/R/P-type hybrid proteins showed a much stronger binding to P1 ori than RepALX15. Activation in Vivo of Replication Origins by Hybrid Proteins To examine whether these hybrid RepA proteins activate an Rts1 ori plasmid in trans, the pTW22:Rts1ori plasmid was introduced into the polA strain JG112 harboring pGB2 recombinant plasmids containing the hybrid repA genes. If the ori activation by RepA was inefficient, as was observed with Rts1 ori plasmid activated by P1 RepAwt, transformant colonies that developed on plates containing both ampicillin and spectinomycin were small in size and number. In addition, such an ori plasmid

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FIG. 4. Binding of hybrid proteins to P1 ori. The 0.30-kb EcoRI–HindIII fragment, end-labeled with 32P (0.05 pmol DNA, 105 cpm), was mixed with fraction II extracts containing the hybrid protein. The experimental procedure was the same as that described in the legend to Fig. 3.

was very unstable. After 5 h of incubation without the antibiotic less than 1% of the cells contained the ori plasmid. RepALX153 was as inefficient as P1 RepAwt in activating the Rts1 origin, but RepALX15 activated it as strongly as Rts1 RepAwt. RepALX151 and RepALX152 activated the origin with intermediate efficiency (Table 2).

Contrary to the Rts1 ori activation, the function of the hybrid proteins toward P1 ori was distinct. Among the P/R/P hybrid proteins, only RepALX153 showed strong activation of P1 ori, and RepALX151 and RepALX152 showed no function in vivo (Table 2). Thus, RepALX152 that contains a small stretch of Rts1 RepA, residues 145–176, in P1 RepA lost the

TABLE 2 a

ori Activation with Hybrid RepA Supplied in trans No. of transformants/mg of DNA

% of colonies with ori plasmidb

Resident plasmid

RepA protein

pTW22:Rts1ori

pBR322:P1ori

pTW22:Rts1ori

pBR322:P1ori

pGB2 pBR322 pALA619 pGB:LX15 pGB:LX151 pGB:LX152 pGB:LX153 pGB:LX17 pGB:LX01

No No P1 RepA RepALX15 RepALX151 RepALX152 RepALX153 RepALX17 Rts1 RepA

,1 3 10 ,1 3 10 6.6 3 102c 5.0 3 104 5.0 3 104c 2.0 3 104c 9.0 3 102c 5.4 3 104c 1.2 3 105

,1 3 10 ,1 3 10 2.6 3 105 3.2 3 102c 1.2 3 102c 1.0 3 102c 4.5 3 104 ,1 3 10 ,1 3 10

,1 95 10 7 ,1 35 95

85 ,1 ,1 ,1 80

a

ori activation by hybrid RepA in trans was examined by transforming plasmid pTW22:Rts1ori or pBR322:P1ori into E. coli strain JG112 (polA) harboring hybrid RepA-producing plasmids pGB:LXn. Results are averages from multiple experiments. b Five single transformant colonies were individually examined for stability of the donor plasmid after 5 h of incubation at 37°C without drug (see Materials and Methods). c A lot of small colonies were found and counted.

DOMAINS OF Rts1 AND P1 RepA

function to activate P1 ori and instead acquired the function to induce moderate activation of the Rts1 origin. It should be noted that RepALX152 retained a strong binding capability to the P1 origin, as shown in Fig. 4. DISCUSSION Our recent study revealed that P1 RepAwt bound in vitro to the Rts1 origin as strongly as Rts1 RepAwt. However, activation in vivo of Rts1 ori by P1 RepAwt was inefficient (Li et al., 1997). In this study we wished to determine which Rts1 RepA subregion can induce a stable ori activation by replacing a portion of P1 RepA with the corresponding portion of Rts1 RepA. As demonstrated, RepALX152 that contains Rts1 RepA residues 145–176 increases the transformation frequency and the stability of the Rts1 ori plasmid in a polA E. coli host markedly as compared with that activated by P1 RepAwt. This subregion of Rts1 RepA is adjacent to residues 177–206, which we had tentatively assigned as an important region for Rts1 ori activation (Tabuchi et al., 1995). In that study, we used a series of hybrid RepA proteins consisting of the N-terminal portion from Rts1 RepA and the C-terminal portion from P1 RepA (R/P-type hybrid). Although we have no explanation for this discrepancy, the present study using the P/R/P-type proteins appears better able to determine such a subregion of Rts1 RepA. In addition, it is interesting that the same region of P1 RepA, residues 145–176, is important for stable activation of the P1 origin, since, although RepALX152 was incapable of P1 ori activation, RepALX153 that contains this P1 RepA subregion, residues 145–176, activates P1 ori as efficiently as P1 RepAwt. Thus, a region required for stable activation of the replication origin was assigned on the P1 RepA as well as on the Rts1 RepA molecule. An important feature is that this subregion is distinct from the regions required for in vitro binding to the origin. Binding required the region between residues 113 and 144 of P1 RepA and the C-terminal region, as shown in the binding study in vitro. It appears that both subregions are needed for efficient binding to P1

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ori. As reported previously (Tabuchi et al., 1995), the region of Rts1 RepA essential for Rts1 ori binding was located between residues 113 and 129. In that study RepAX17 (Rts1 RepA 113/P1 RepA 174) showed no binding but RepAX16 (Rts1 RepA 129/P1 RepA 158) could bind, though weakly, to Rts1 ori. Also, RepAX15 (Rts1 RepA 145/P1 RepA 142) bound to the origin as strongly as Rts1 RepAwt without showing an evident activation of the origin in vivo. These findings suggest that the region between residues 113 and 144 of Rts1 RepA is enough for efficient binding to Rts1 ori, while two regions of P1 RepA are required for binding to P1 ori. As the region for stable ori activation is quite specific for each plasmid, the region may not be a site that interacts with common host factors such as DnaJ, DnaK, and GrpE proteins which are known to be required for activation of P1 RepA to induce replication (Sozhamannan and Chattoraj, 1993; Chattoraj et al., 1996). Unlike the initiator proteins of pT181, a specific endonuclease (Dempsey et al., 1992; Projan and Novick, 1988; Wang et al., 1993), and ColE2, an RNA primase (Takechi and Itoh, 1995), both P1 RepA and Rts1 RepA have no known enzymatic activity. Accordingly, the specific mechanism of the ori activation region is unknown. A possibility is that the region directs a correct contact of RepA molecules with ori iterons. In other words, the region may be involved in forming a functional mature complex of RepA– DNA leading to the efficient initiation of replication, as proposed by Brendler et al. (1997). In this context, it is noteworthy that the Rts1 RepA mutations, copI (R142K) (Kamio et al., 1984) and copA (H159N) (Yonemitsu et al., 1995), which both induce the high copy state, are located close to or within this subregion. The DNA binding domain assigned to the Rts1 RepA and P1 RepA molecules might be involved in an initial contact with origin sequence. Interestingly, this region corresponds to domain II (Rts1 RepA residues 112–131 and P1 RepA residues 111–130), which is proposed by Gibbs et al. (1993) as the DNA binding domain in Rts1 RepA and P1 RepA.

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ACKNOWLEDGMENTS We thank Ann L. Abeles for critical reading of the manuscript and for anti-P1 RepA antibody. We also thank Akira Tabuchi, Makoto Ohnishi, Takahiro Murata, and Qing-Bao Tian for valuable discussions and Kaori Sato for her technical assistance. This work was supported by a Grant-in Aid for Scientific Research (07457072) from the Ministry of Education, Science and Culture of Japan and by grants from the Yakult Foundation and the Aiko Foundation.

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