©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Nicking-Closing Activity of the Initiator of Replication Protein RepB of Plasmid pMV158 on Supercoiled or Single-stranded DNA (*)

(Received for publication, August 8, 1994; and in revised form, November 2, 1994)

Miriam Moscoso Gloria del Solar Manuel Espinosa (§)

From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Asymmetric rolling circle replication of the promiscuous replicon pMV158 is initiated by the plasmid-encoded RepB protein. In vitro, purified RepB protein introduces a nick within the leading strand origin of replication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide were recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 °C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able to nick (initiation) and to covalently close (termination) single-stranded oligonucleotides containing the protein cleavage sequence. Covalent DNA-protein complexes were not found, indicating that RepB has unique features among plasmid-encoded proteins involved in rolling-circle replication or conjugative mobilization.


INTRODUCTION

Single-stranded coliphages and many small multicopy plasmids replicate through an asymmetric RC (^1)mechanism, which is characterized by the uncoupling of the leading and lagging strand synthesis (Baas and Jansz, 1988; Novick, 1989; Gruss and Ehrlich, 1989; del Solar et al., 1993a). The current model for plasmid leading strand replication is mainly based on pT181 and related plasmids (Rasooly and Novick, 1993; Rasooly et al., 1994) and involves various stages: (i) a dimer of the plasmid-encoded initiator of replication (Rep) protein binds to the plasmid DNA at a specific region in the double strand origin, dso (Wang et al., 1993); (ii) one Rep subunit cleaves the plasmid (+) strand by introducing a thyrosil phosphodiester covalent bond at a specific DNA sequence (Thomas et al., 1990); (iii) the Rep-generated nick leaves a free 3`-OH end, which is extended by host proteins that use the(-) strand as a template and displace the parental nicked (+) strand; (iv) the replisome reaches the reconstituted dso and extends several more nt; and (v) the newly synthesized strand is closed by the unused Rep monomer through at least three nucleophylic DNA-protein-DNA attacks, leaving a short oligonucleotide covalently bound to the attacking Rep monomer. Thus, the final DNA products of plasmid leading-strand replication are one dsDNA molecule and one ssDNA intermediate (te Riele et al., 1986). One distinctive feature of this model is that the Rep protein cannot be reused after each round of replication since the final protein configuration is a heterodimer composed of an intact Rep moiety and an inactive Rep-oligonucleotide subunit (Rasooly and Novick, 1993).

Plasmid pMV158 is a 5536-bp multicopy natural promiscuous replicon that is the prototype of a family of RC-plasmids isolated from several eubacteria (del Solar and Espinosa, 1992; del Solar et al., 1993a). Various genes have been identified as being involved in conjugative mobilization (mob), tetracycline resistance (tet), and replication and its control (repB, copG, rnaII; Fig. 1). This latter region has been characterized in some depth for plasmid pLS1 (a Deltamob derivative of pMV158; del Solar et al., 1993a, and references therein). The Rep proteins of the pMV158 family share conserved motifs with plasmid-encoded Tra and Mob proteins as well as with Rep proteins of RC-plasmids, ss coliphages, and geminiviruses (Pansegrau and Lanka, 1991; Ilyina and Koonin, 1992). However, the DNA sequence at the dso of the pMV158-plasmid family has no known homology with other replicons (del Solar et al., 1993a). Initiation of pMV158 replication is mediated by RepB protein (24.2 kDa), which has been shown to introduce a specific nick between nt 448 (G) and 449 (A) of the complete nucleotide sequence of pMV158 (de la Campa et al., 1990, Priebe and Lacks, 1989). Supercoiled DNA is the only productive substrate for replication of pMV158 in a cell-free extract (del Solar et al., 1987), and the nick site is located on the loop of one of the major plasmid secondary structures, Hairpin I (Puyet et al., 1988). This Hairpin I is located within a region (termed nic) physically distant of the RepB-binding site (del Solar et al., 1993a). In vitro, RepB protein binds to a set of three directly repeated sequences or iterons (the bind region) located 84 bp downstream of the nick site (de la Campa et al., 1990; del Solar et al., 1993a).


Figure 1: Features of pMV158. A, transcription map showing promoters (triangles), mRNAs, and antisense RNAs (wavylines) with the direction of transcription indicated (arrowheads). The region missing in pLS1 is indicated. The origins of leading (dso) and lagging (sso) strands are shaded. Synthesis of RepB is regulated at the transcriptional level by the copG gene product. Both copG and repB are co-transcribed from promoter P. CopG binds to a plasmid region, which includes the -35 region of P, thus repressing its own synthesis and synthesis of RepB (del Solar et al., 1990). At the translational level, the amount of productive repB mRNA could be regulated by the binding of the antisense RNA II to a region in the cop-rep mRNA, which includes the repB initiation of translation signals (del Solar and Espinosa, 1992). B, the minimal dso of pMV158 as defined by deletion analysis. Right and leftborders of the regions missing in plasmids pLS1Delta24 (rightborder) and pLS1DeltaA4 (leftborder) are indicated. Positions of Hairpin II, Hairpin I, and the RepB-nick site () are also shown. The RepB-generated footprints (bullet), determined on linear dsDNA, are indicated on both strands. Downstream of the nick site, the three 11-bp iterons (boldface, underlined) are located. These iterons are the in vitro determined RepB-binding site (de la Campa et al., 1990). Other indicated features are the promoter for copG and repB genes (P), the initiation site of the cop-rep mRNA, the copG Shine-Dalgarno sequence (underlined), and the CopG initiation codon (boldface). C, nucleotide frequency distribution at the dso of pMV158 between coordinates 400 and 500. Note that the RepB-nick site (arrow) lies within a G+C-rich central region, which is flanked by sequences with high A and T content, suggesting that this region has a high potential to generate secondary structures.



In the present work we show that, in vitro, purified RepB protein relaxed supercoiled pMV158 DNA in a topoisomerase I-like reaction, having an optimal activity at 60 °C. The protein was also able to nick and to covalently join two ss oligonucleotides containing the RepB-nicking sequence. These reactions reproduce initiation (nicking) and termination (closing) of RC replication. In addition, we have defined the bases involved in the cleavage mediated by RepB. On linear DNA, RepB absolutely required the nicking region to be in a single-stranded configuration. We have been unable to isolate any RepB-oligonucleotide covalent intermediates, and filter-binding assays showed that the RepBbulletDNA complexes were sensitive to treatment with SDS. We propose that a RepBbulletDNA association, different than a stable covalent linkage, is involved in the plasmid initiation of replication, similar to that of the gpII protein of coliphage fd (Meyer and Geider, 1979). This is the first example of a plasmid-encoded protein able to catalyze a site-specific nicking reaction without forming a stable protein-DNA covalent linkage.


MATERIALS AND METHODS

DNA and Oligonucleotide Purifications

Supercoiled plasmid pMV158 DNA was isolated from cultures of Streptococcus pneumoniae by preparation of cleared lysates followed by two consecutive CsCl/ethidium bromide equilibrium gradients, as reported (del Solar et al., 1987). Oligonucleotides were purified essentially as described (Sambrook et al., 1989). In some cases, the oligonucleotides were not deprotected from tritylation after synthesis. In these cases, purification was performed by employing oligonucleotide purification cartridges (Applied Biosystems), following the instructions of the vendor. In other cases, oligonucleotides were further purified by HPLC.

Purification of RepB

RepB protein was purified from Escherichia coli BL21DE3 (a gift of B. Studier) harboring plasmid pLS19 (del Solar et al., 1990), essentially as described (de la Campa et al., 1990) except that the DEAE-Sephacel step was omitted. The concentration of the protein preparation used here was 45 ng/µl, as calculated from its molar extinction coeffient and from determination of its amino acid composition. The protein was stable for more than 1 year at -70 °C. Analytical ultracentrifugation experiments were performed with two concentrations of RepB (in 20 mM TrisbulletHCl, 1 mM EDTA, and 0.2 M KCl) at two temperatures, 10 and 25 °C. Sedimentation values were corrected for the specific volume of the protein (157.31 ml/g), and for the molecular mass value of the RepB monomer (24,252 Da, as deduced from the DNA sequence). The final calculations gave a molecular mass of the RepB preparation of 6.056, that is a hexamer. Details of sedimentation data will be published elsewhere.

Labeling of the Oligonucleotides

Labeling of the oligodeoxyribonucleotides at their 3`-ends was done by using [alpha-P]ddATP (5000 Ci/mmol) and terminal deoxynucleotidyltransferase; labeling at the 5`-ends was performed by using [-P]ATP (5000 Ci/mmol) and bacteriophage T4 polynucleotide kinase (Amersham Corp.). Procedures for labeling followed published protocols (Sambrook et al., 1989).

Assays of RepB Activity on Supercoiled DNA

Under standard conditions, mixtures of RepB protein (45 ng) and pMV158 DNA (700 ng) were incubated in a total volume of 30 µl of buffer NB (20 mM TrisbulletHCl, pH 8.0, 1 mM EDTA, 100 mM KCl, 5 mM dithiothreitol, 20 mM MnCl(2)) for 30 min at the temperatures indicated in the text. In some cases, the nicking-closing reactions were performed in the presence of nicotinamide mononucleotide to control the possible presence of contaminant-adenylated DNA ligase in the RepB preparation (Lehman, 1974). After incubation, samples were treated with Proteinase K (125 µg/ml), incubated at 20 °C for 10 min, and stopped by the addition of the loading buffer. The same results were obtained when Proteinase K was omitted, and the reactions were stopped by the addition of 0.25 M EDTA. Reaction products were analyzed by electrophoresis in 1% agarose gels with ethidium bromide (0.5 µg/ml) in 90 mM Tris borate, 2 mM EDTA buffer. The amount of specifically relaxed DNA was determined by laser densitometry of various negatives obtained from agarose gels. Since open circular (FII), relaxed covalently closed (FI`), and supercoiled (FI) plasmid forms give different specific fluorescence intensities, the yield of RepB nicking-closing products was calculated from the decrease of the fluorescence signal of the FI form relative to that of the untreated DNA. Treatment of supercoiled DNA with the KCl-containing incubation buffer resulted in some unspecific generation of FII forms.

Nicking Activity of RepB on Single-stranded Oligonucleotides

Unless otherwise stated, labeled oligodeoxyribonucleotides (3.75 pmol) were incubated with RepB (at the concentrations indicated under ``Results'') in 90 µl of NB for 30 min at 37, 45, or 60 °C. Reactions were stopped by the addition of Proteinase K (320 µg/ml), and samples were incubated for an additional 20 min at 37 °C. The reaction products were separated on 20% polyacrylamide sequencing gels containing 8 M urea. Radioactive bands were detected by autoradiography on Kodak X-Omat films, and the counts in the bands of the gels were directly quantified with the PhosphorImager ImageQuant equipment (Molecular Dynamics).

Nicking-Closing Reactions

Labeled oligodeoxyribonucleotides (1.3 pmol) were mixed with unlabeled ones (2.5 pmol) and incubated with RepB (29 or 56 ng, 1.2 or 2.3 pmol, respectively) in 30 µl of NB for 30 min in the above conditions. Samples were treated, electrophoresed, and quantified as above.

Filter-binding Assays

The 23-mer oligodeoxyribonucleotide 5`-GGGGGGGCTACTACGACCCCCCC-3`containing the wild-type nic region of pMV158 was labeled at its 3`-end and incubated with RepB protein in the nicking conditions (see above) at 60 °C. Reaction mixtures were loaded onto nitrocellulose filters (Millipore HA 0.45 µm) presoaked with NB buffer containing a 10-fold excess of a 23-mer cold unspecific oligonucleotide. Samples were filtered at a flow rate of about 1.5 ml/h, and filters were washed with 9 ml of NB buffer at the same flow rate. To analyze the stability of the RepBbulletDNA complexes, filters were washed with either NB to which the pH was adjusted to a range between 7.0 and 9.5 or NB-containing various amounts of KCl, NaCl, or SDS. Filters were dried, and the retained radioactivity (Cerenkov) was determined. Control samples did not receive RepB, and their value (about 80 cpm) was subtracted from each sample. Each experiment was performed at least twice.


RESULTS

In Vitro Activity of RepB Protein on Supercoiled pMV158 DNA

The minimal functional dso of pMV158 has been defined in vivo as located within a 247-bp region, which includes the three iterons and the RepB nick site (del Solar et al., 1993b; Fig. 1). Filter-binding assays with linear dsDNA showed that RepB protein specifically binds to a fragment that contains the bind region and did not bind to another DNA fragment containing the nic region (de la Campa et al., 1990). In addition, DNase I and hydroxyl radical footprintings on linear dsDNA with purified RepB protein (de la Campa et al., 1990), showed that the RepB-protected region encompasses the three iterons, the footprints not extending further toward Hairpin I (Fig. 1B). The frequency and distribution of nt around the plasmid nic region (Fig. 1C) shows a G+C-rich sequence surrounding the RepB nick site (448/449), flanked by A+T-rich sequences, a feature which is considered important in the generation of cruciform structures (Bowater et al., 1991). Hairpin I has been mapped on supercoiled pLS1 DNA by sensitivity to nuclease S1 (Puyet et al., 1988). It would thus seem that exposure of the unpaired nick site to RepB is an important stage in the initiation of pMV158 replication.

We had observed that the incubation of supercoiled pLS1 DNA with purified RepB protein at 37 °C resulted only in partial relaxation of the plasmid, even at high protein/DNA ratios or prolonged incubations (de la Campa et al., 1990). Plasmid pLS1 replicates in the mesophyle Bacillus subtilis, and a moderate increase in its copy number has been observed when the cells are grown at 45 °C instead of at 37 °C. (^2)We incubated supercoiled pMV158 DNA with purified RepB protein at 37, 45, and 60 °C (Fig. 2A). Treatment of supercoiled (form FI) DNA with RepB specifically yielded a mixture of nicked (FII) and covalently closed relaxed (FI`) plasmid molecules, which reflect, respectively, the nicking and nicking-closing ability of RepB. Generation of forms FII+FI` was RepB-dependent, since only a slight amount of such forms was observed in samples not treated with the protein (Fig. 2A). To rule out the possibility that some contaminant adenylated DNA ligase was present in the RepB preparation (which could account for the closing reaction; Lehman, 1974; Kornberg and Baker, 1992), plasmid DNA was incubated with various amounts of nicotinamide mononucleotide in the presence or absence of RepB. The results (Fig. 2A) showed no significant influence of this compound on the nicking-closing reaction, which allows us to conclude that RepB is able to relax and to reseal supercoiled pMV158 DNA. At a fixed time, and at a constant concentration of RepB and its substrate, the amount of FII+FI` DNA increased with the temperature, varying between 60% (at 37 °C) and 95% (at 60 °C). At 32 °C or below, less than 25% of the input DNA was affected by incubation with RepB (not shown). A time-course experiment showed that the RepB-mediated nicking (and nicking-closing) was very fast at 60 °C, since about 80% of the substrate DNA was converted to FII+FI` molecules after 1 min of incubation (Fig. 2B). The reaction slowed down at 45 °C, and no plateau was reached at 37 °C. Concentration dependence assays (Fig. 2C) indicated that a majority of the pMV158 DNA was relaxed by RepB in a period of 30 min in the presence of 34 ng of the protein (or below, depending upon the temperature). Similar results were obtained when supercoiled pLS1 plasmid DNA was used as substrate (not shown). In the above experiments, the ratio of RepB molecules to DNA molecules was 9.5:1, assuming that all protein molecules in our RepB preparation were active, and that the active species of the protein is a monomer. However, analyses of the RepB-configuration by analytical ultracentrifugation showed that RepB is an hexamer (not shown). The activity of RepB depended on the presence of Mn, this cation being partially replaceable by Mg (de la Campa et al., 1990). Incubation with the topoisomerase I-inhibitor camptothecin (Avemann et al., 1988), or with the pMV158-encoded CopG protein (del Solar et al., 1990), did not affect the RepB-mediated reactions (not shown). Relaxation of supercoiled DNA by RepB did not seem to require high energy cofactors, since incubation with ATP did not influence the reactions. No consensus ATP-binding domain has been found in RepB.


Figure 2: Nicking of pMV158 supercoiled DNA by purified RepB protein. A, supercoiled pMV158 DNA (700 ng) was incubated or not with purified RepB protein (45 ng; protein/DNA ratio of 9.5:1 in molecules), in the presence or absence of the indicated amounts of nicotinamide mononucleotide (BNMN) at the indicated temperatures for 30 min. Samples were run in agarose gels with ethidium bromide. Gels were photographed, and the negatives were scanned to quantify the results. The main observed plasmid forms are supercoiled (FI), open circular (FII), and closed relaxed circles (FI`). Note that the RepB-untreated samples contained some FII forms. (+) and(-) indicate the presence or absence of protein, respectively. M, molecular weight standards. B, time-course of the nicking-closing reaction on supercoiled pMV158 DNA by purified RepB protein. C, concentration-dependence of the nicking-closing reaction on supercoiled pMV158 DNA (700 ng/sample) by purified RepB protein. Reactions were performed at 37 (), 45 (up triangle), and 60 °C (bullet).



RepB-nicking Activity on Single-stranded Oligonucleotides

We next assayed the in vitro activity of RepB protein on single-stranded oligonucleotides. To this end, a 23-mer oligonucleotide containing the RepB wild-type nick site (see Fig. 4, oligo wt) was synthesized, labeled at its 5`-end, and treated with increasing amounts of RepB at the three above mentioned temperatures. The reaction products were separated by electrophoresis on a 20% sequencing gel. If RepB were able to nick this substrate, a labeled 15-mer product should be observed. The results (Fig. 3A) showed that a band of this size was generated in all of the RepB-treated samples, its intensity being increased as the ratio of RepB/DNA was augmented. Several labeled bands were visible in the untreated samples, some of them sensitive to RepB-nicking. Purification of the labeled 23-mer oligonucleotide by HPLC did not improve this pattern; extraction of the upper 23-mer band from native polyacrylamide gels resulted in the failure of RepB to act on this substrate. We assume that these bands are due to the G(7) residues located at the 5`-end of the 23-mer, which could either generate intrastrand pairings with the C(7) nt or complicate the oligonucleotide synthesis. To avoid these inconvenients, a second 23-mer oligonucleotide containing the RepB-nick sequence, but lacking any possible intrastrand pairing (Fig. 4, oligoC), was synthesized and treated with RepB. In this case, a clear band of 15-mer, resulting from RepB-cleavage of the input ssDNA was observed (Fig. 3B). Quantification of the reaction products showed that the RepB-concentration dependence curves resembled those observed for supercoiled DNA (Fig. 3C). The single-stranded 23-mers appeared to be slightly more sensitive to RepB than the supercoiled DNA, since 90% digestion of the substrate (60 °C, 30 min) was achieved at the protein/DNA ratio (in molecules) of 3:1 for ssDNA and of 9.5:1 for supercoiled DNA. The results demonstrate that RepB is able to digest single-stranded DNA substrates, having or not having a possible intrastrand pairing, but sharing the sequence: 5`-TACTACGAC(6)-3`.


Figure 4: Sequence specificity of RepB. The activity of purified RepB protein was assayed either on a 23-mer single-stranded oligonucleotide containing the wild-type (wt) pMV158-nic region (5`-G(7)CTACTACG/AC(7)-3`) or on 23-mer having the indicated base changes (boxed). The oligonucleotides (1.3 pmol) were labeled at its 5`-end with T4 polynucleotide kinase and were incubated with RepB protein (9.3 pmol) for 30 min at 37, 45, and 60 °C. Protein/DNA ratio (in molecules) was 7.3:1. Reaction products were separated on sequencing gels and directly quantified. The amount of nicked DNA in each case was compared with that obtained for the wt oligonucleotide. Oligonucleotide comp contains the nucleotide sequence complementary to the wt. A ds oligonucleotide made by hybridization of the wt and the comp 23-mers was not nicked by RepB (not shown).




Figure 3: Nicking activity of RepB on ssDNA. A, the 23-mer single-stranded oligonucleotide containing the wild type nic region of plasmid pMV158 (oligowt: 5`-GGGGGGGCTACTACGACCCCCCC-3`) was labeled at its 5`-end with T4 polynucleotide kinase. 2.5 pmol of the labeled oligo was treated with 9.3 pmol of RepB (ratio protein/DNA of 3.7:1, molecules) at the indicated temperatures. The upper band (arrow) is the 23-mer oligonucleotide, the band below it (arrow) and the other bands are, most likely, products from defective oligonucleotide synthesis or of intrastrand pairing. They are also cleaved by RepB. The lowerband is the 15-mer cleavage product, which co-migrates with a labeled band. B, the 23-mer single-stranded oligonucleotide 5`-TGCTTCCGTACTACGACCCCCCA-3` (oligoC, Fig. 4), which lacked any possible intrastrand pairing, was 5`-end labeled and treated with RepB, as in panelA, except that the amount of RepB was increased to show maximum nicking. Note that no bands migrating between the 23- and the 15-mer products are visible in this case. M, ladder marker. C, The labeled oligo wt 23-mer (3.7 pmol) was incubated during 30 min, at 37 (), 45 (up triangle), and 60 °C (bullet) with various concentrations of RepB (3.7, 7.5, 11.2, 15, 18.5, and 28 pmol). The protein/DNA ratios (in molecules) were of 1:1, 2:1, 3:1, 4:1, 5:1, and 7.5:1, respectively. As controls, RepB-untreated samples were run. The band co-migrating with the 15-mer RepB-product was considered as control and subtracted from the RepB-treated samples after direct quantification of the RepB products.



The sequence specificity of RepB on the nick site was tested by assaying its nicking activity on single-stranded 23-mer oligonucleotides in which base changes at the RepB-nick region were introduced. The reaction products were quantified and compared with the RepB nicking activity on the 23-mer containing the wild-type nick site. As can be observed (Fig. 4), at 5` of the nick site (G/A) only a GA change was allowed, although it reduced by 50% the amount of digested DNA. At 3` of the nick, the A could be changed by C or T without much reduction in the RepB activity. However, one G at this position made the oligonucleotide insensitive to RepB. In addition, a C to G change in the second nt after the nick site (CG) resulted in a substrate not recognized by RepB. This finding was unexpected because we had ascribed the C(7) tract at 3` of the nick site as being mainly involved in the generation of Hairpin I on supercoiled DNA, by intrastrand pairing with the G(7) tract 5` of the nick. Generation of this structure on supercoiled DNA would indeed expose the phosphodiester bond in the GpA nick site as a single-stranded region. The failure of RepB to nick the 23-mer containing the CG change, could indicate either a requirement for the generation of a secondary structure in the ssDNA substrate or that the nt at this particular position is important. The latter explanation is more likely, since (i) the oligo B (having the CG change) could generate a secondary structure but was still insensitive to RepB and (ii) the oligo C is fully sensitive to RepB and yet it shuld not generate any intrastrand pairing. In addition, changing the C-stretch downstream of this C nt by A and T residues did not affect cleavage by RepB (not shown). The protein did not recognize the sequence complementary to its nicking region (oligo comp), which demonstrates that nicking by RepB is sequence-specific. This oligonucleotide also contains the G(7) and C(7) ends, so that intrastrand pairings and secondary structures within the RepB substrate are not important features when the substrate is in a single-stranded configuration. We conclude that C also plays an important role within the DNA sequence for RepB nicking. The critical role of sequences downstream of the nick site for RepB-cleavage contrasts with the requirements observed for TraI protein of plasmid RP4, which requires 6 nt upstream of the nick site but has no base specificity downstream of it (Pansegrau et al., 1993). Finally, a ds oligonucleotide containing the wild type nicking region, obtained by hybridization of the oligonucleotides wt and comp, was not a substrate of purified RepB protein (not shown). Since RepB relaxed supercoiled DNA (Fig. 2) but did not cleave linear dsDNA, it would appear that the nic region has to be exposed in a single-stranded configuration within the supercoiled DNA plasmid molecules. In fact, RepB was unable to nick linear ds pLS1 DNA as judged from alkaline agarose gels. (^3)RepB protein specifically cleaved ssDNA harboring some changes in the nic region (Fig. 4), suggesting that the protein could recognize as substrate other dso of pMV158-related plasmids. We have preliminary evidence suggesting that this is, indeed, the case. We can then draw the following conclusions: (i) the DNA sequence cleaved by RepB has to be in a single-stranded conformation; (ii) generation of a secondary structure within the 23-mer is not required; and (iii) the protein recognizes the 9-nt consensus sequence (5`-TACTACR/HC (IUB code, R = puryne; H = not G). A computer search revealed that no other nt sequence of this kind is present in pMV158.

RepB Is Able to Seal the Nick

The above results show that RepB has the ability of nicking ssDNA in a site-specific reaction. Consequently, the protein should be able to initiate rolling circle replication. Termination by this replication mechanism has to be achieved by (i) the introduction of a new nick, (ii) closing of the nick on the newly synthesized strand, and (iii) the covalent joining of the ends of the displaced strand in a reaction probably catalyzed by RepB. The protein is able to nick and then to close supercoiled DNA as judged from the specific generation of forms FI` (Fig. 2), but details of the closing reaction are more difficult to analyze with this substrate. To know how the RepB protein would seal the nick it generated, two oligonucleotides of different sizes (23- and 26-mer), and both having the nick site located at different distances from their ends, were synthesized (Fig. 5). Either one or the other of the two oligonucleotides was 5`-labeled, mixed with twice the amount of the other unlabeled oligonucleotide, and treated with RepB protein. If a RepB-mediated cleavage-joining reaction occurred, a new band corresponding to a labeled 29-mer DNA product should appear when using the labeled 23-mer. Conversely, the reaction product that should be detected when the labeled ssDNA was the 26-mer is a new band corresponding to a 20-mer oligonucleotide. As can be seen (Fig. 5), bands corresponding to the products of nicking and nicking-closing reactions were observed in the RepB-treated samples, similar to the results found for TraI protein of plasmid RP4 (Pansegrau et al., 1993).


Figure 5: Nicking-closing activity of RepB on ssDNA. The indicated 23- and 26-mer single-stranded oligonucleotides were labeled at their 5`-ends. Both oligonucleotides contain the RepB nick site (/) in a different position. Mixtures of labeled and unlabeled substrates were made and used as a substrate for RepB protein. For each reaction, the protein DNA ratio (in molecules) was of 0.3:1 or 0.6:1. The assays were performed at the indicated temperatures. The labeled nicking and nicking-closing products were visualized on sequencing gels; M, labeled oligonucleotide mixtures used as markers. The expected reaction products are indicated on the top of the figure.



The closing reaction mediated by RepB was further analyzed by attempts to join oligonucleotides that contained, respectively, the right and the left half of the nick region (Fig. 6). First, a 5`-labeled 15-mer ending at the nick site (pG-OH) was mixed with a phosphorylated 8-mer initiating at the nick site (pAp). If RepB were able to covalently join both halves of its nick region without previous nicking, a 23-mer reaction product should be detected. The results (Fig. 6A) showed that this was not the case, indicating that the substrate has to be cleaved by the protein prior to the closing reaction. To test this, the same labeled 15-mer (ending in pG-OH) was mixed with a labeled 26-mer oligonucleotide, which contains all of the wild-type nicking region, and treated with RepB (Fig. 6B). In this case, the protein cleaved the 26-mer to generate a labeled 12-mer product. In addition, a new band corresponding to a labeled 29-mer oligonucleotide was also identified. The new 29-mer product should arise from the RepB-mediated covalent join of the nicked 3`-half (14-mer) with the 3`-OH end of the 15-mer. This closing reaction was not observed if the 15-mer to be joined has an end different than the wild-type, even if the pG-OH was kept unchanged (not shown). A complementary experiment was performed; the above 26-mer was mixed with a labeled 8-mer containing the region 3` to the nick and was incubated with RepB. The 20-mer closing product was not detected (Fig. 6C). Taking these two results together, we conclude that the protein can only close the nick if a RepB-generated 5`-phosphate end is provided. The 3`-OH end to be closed has to belong to the nick sequence, but it does not need to be generated by the protein. These results indicate that the RepB-mediated nicking reaction would leave an ``activated'' 5`-phosphate end 3` to the nick. Since RepB failed at nicking oligonucleotides extracted from polyacrylamide gels, verification of this putative activation by reisolation of the 14-mer RepB-generated intermediate (Fig. 6B) was not feasible. In addition, we have observed that gel retardation assays performed with RepB and the 26-mer oligonucleotide did not generate any retarded band (not shown), perhaps indicating that protein-DNA complexes were dissociated during electrophoresis. Whether ``activation'' is the consequence of a reaction that requires the simultaneous presence of the protein and the two substrates to be joined (the 26- and the 15-mer, Fig. 6B) and whether this reaction could be somehow uncoupled is under investigation.


Figure 6: RepB-mediated closing reactions. Schematic representations of the nicking-closing experiments performed with single-stranded oligonucleotides, and the expected results after RepB-treatment are depicted at the top of each panel. The reaction products are shown at the bottom. The substrates employed were the oligonucleotides indicated in Fig. 5, labeled at their 5`- or 3`-ends (indicated by *) or unlabeled. The oligonucleotides were treated (+) or untreated(-) with RepB at the indicated incubation temperatures. The lengths of the detected oligonucleotides are indicated at the sides of the different autoradiograms. A, the 5`-labeled 15-mer, ending in pG-OH, was mixed with the unlabeled 8-mer starting in pA. Note that no 23-mer closing product is observed. B, the 5`-labeled 26-mer containing the GpA RepB-nicking site was mixed with the labeled 15-mer ending in pG-OH. Note the appearance of the 29-mer RepB-generated product. C, the above 26-mer was mixed with the phosphorylated 5`-labeled 8-mer (starting in pA). The 20-mer closing product was not detected. D, the 23-mer was 3`-end labeled, treated with RepB, and the reaction products were digested with Proteinase K. A mixture of phosphorylated and nonphosphorylated 5`-labeled 8-mer was run; the latter migrates slower than the former. Generation of a tyrosylphosphodiester covalent bond between RepB and its substrate should yield a peptide-DNA adduct having an electrophoretic mobility different from that of the free phosphorylated 8-mer.



Several nicking-closing enzymes (Rep protein of the RC-plasmid pT181, gene A product from X174, and Tra and Mob proteins from plasmids RP4 and RSF1010) are able to nick and close DNA through a stable covalent protein-DNA intermediate (Rasooly and Novick, 1993; Langeveld et al., 1978; Pansegrau et al., 1990; Scherzinger et al., 1992). To know if a covalent RepB-DNA intermediate is generated, the 23-mer oligo wt (Fig. 4) was labeled at its 3`-end and treated with RepB protein. The reaction products were digested with Proteinase K in order to leave a short peptide linked to the DNA if a covalent protein-DNA complex were generated. As can be seen (Fig. 6D), only free DNA species of 8-mer in size (in addition to the 23-mer) were detected. These are the expected products if no covalent DNA-peptide heteromolecules are the reaction products. Several other experiments aimed at detecting any possible RepB-DNA covalently linked intermediates, using either supercoiled DNA or single-stranded oligonucleotides as substrates, gave negative results (not shown). To analyze the nature of the RepBbulletDNA complexes, we made use of a filter-binding assay (de la Campa et al., 1990). To this end, the 23-mer oligo wt was 3`-end labeled and incubated with RepB at 60 °C, in conditions of maximum nicking (Fig. 3). The reaction products were filtered, and the filters were washed with various buffers (Table 1). The results showed that RepBbulletDNA complexes were extremely sensitive to SDS, again supporting the noncovalent nature of the RepB-binding to DNA. Complexes were moderately sensitive to high salts, indicating that electrostatic interactions take place between the protein and its target DNA. The predicted isoelectric point of RepB is 8.8, so we should expect a reduction in the proteinbulletDNA complexes once this point is surpassed. Washing buffers with pH above this point also reduced the amount of DNA retained in the filters (Table 1), suggesting that the interactions between RepB and DNA could take place through positively charged amino acids. Based on all of the above data, we tentatively conclude that RepBbulletDNA interaction can be exerted either through the generation of a transient phosphodiester bond, as is the case with the gene II product of coliphage fd (Meyer and Geider, 1979), or through a noncovalent binding.




DISCUSSION

The initiator of replication RepB protein from plasmid pMV158 has nicking-closing, sequence-specific activities, as expected for a protein involved in RC-replication. We have simulated a process in which purified RepB protein performed the reactions involved in initiation and termination of plasmid RC-replication. RepB has specific nicking activity on ssDNA containing the nic region, which is the one present in the plasmid (+) strand. RC-replication shares features with conjugative processes since ssDNA intermediates are generated, and proteins involved in conjugative transfer also nick their substrate DNA (Pansegrau et al., 1990, 1993). However, in contrast to other Rep, Tra, and Mob plasmid-encoded proteins, which generate a proteinbulletDNA covalent bond (Thomas et al., 1990; Pansegrau et al., 1990; Scherzinger et al., 1992; Rasooly and Novick, 1993), RepB protein of pMV158 did not show any detectable covalent link with DNA. RepB absolutely required single-stranded or supercoiled DNA substrate to nick its target since it failed to cleave a ds oligonucleotide containing the nicking site and it was unable to nick linear ds pLS1 DNA.^3 In addition, replication of pMV158 in a cell-free extract absolutely required supercoiled DNA (del Solar et al., 1987). The RepB-requirement for supercoiled substrate and its activity on ssDNA strongly indicate that generation of Hairpin I and exposure of the nick site in a single-stranded configuration is essential for initiation of replication of pMV158 (del Solar et al., 1987, 1993b, this work). The above features of RepB resemble those found for proteins A and gpII from coliphages X174 and fd respectively, since both proteins also need the substrate to be supercoiled or single-stranded (Langeveld et al., 1978; Meyer and Geider, 1979; van Mansfeld et al., 1980). Although negatively supercoiled DNA seems to be the preferred substrate for nicking-closing proteins, the MobABC complex of plasmid RSF1010 nicks linear dsDNA with identical requirements as those for supercoiled DNA, except that more MobC is needed (Scherzinger et al., 1992). Furthermore, RepC of plasmid pT181 acts on supercoiled DNA (Noirot et al., 1990), but it is able to conduct in vitro replication with a relaxed DNA substrate (Khan et al., 1982) and shows specific nicking activity on lineal dsDNA in vitro (Koepsel and Khan, 1987). The reaction conditions of RepB also differ from other nicking-closing enzymes. RepB is an hexamer; it shows an optimal temperature at 60 °C, and it requires Mn, whereas the other proteins in general have a dimeric configuration, they require Mg and show optimal temperature for cleavage at 37 °C or below.

Many of the Tra and Mob proteins exhibit common motifs that are also conserved in the Rep proteins of RC plasmids and ssDNA coliphages (Pansegrau and Lanka, 1991; Ilyina and Koonin, 1992; Waters and Guiney, 1993). Furthermore, the dso of many RC-plasmids, the (+) origins of ssDNA coliphages, and the oriT of conjugative plasmids have sequence homologies at their nic region (Waters and Guiney, 1993). Although the protein motifs are conserved in the pMV158 plasmid family, the nic region in the DNA of these plasmids has no resemblance with known cognate regions in other plasmids or coliphages (del Solar et al., 1993a). Three possible Tyr residues (at positions 14, 99, and 115) could be the candidates for realizing the nucleophilic attack on the phosphodiester bond at the RepB nick site by a tyrosil-OH group. The residue most likely involved in this reaction is Tyr, since it is conserved among all nicking-closing proteins from plasmids and viruses (Ilyina and Koonin, 1992).

One feature of the plasmids of the pMV158 family is that they all contain two or three iterons of different lengths, which are separated from the nick site by a spacer sequence ranging between 13 and 91 bp (del Solar et al., 1993a). In the case of pMV158, purified RepB protein binds to the iterons and introduces the nick 84 bp upstream of its binding site (de la Campa et al., 1990). Nevertheless, with the substrates employed here, RepB was able not only to nick and close pMV158 supercoiled DNA but also linear ssDNA, which lacked the iterons. This behavior differs from gpII protein of the filamentous phage f1, which requires the presence of its binding region (in a ds configuration) to introduce the nick within a single stranded oligonucleotide (Higashitani et al., 1994). Although the protein did not generate a stable covalent linkage to DNA, the 5`-end (but not the 3`-end) generated in the nicking reaction was activated by RepB. The ability of RepB to join the 5`-phosphate end of the nicking reaction product with a new 3`-end (Fig. 6B), in conjuction with the failure of the protein to catalize the joining of the 3`-OH generated during nicking with a provided 5`-phosphate end (Fig. 6C), indicated that the 5`-end to be closed is activated somehow by the RepB nicking. This RepB-mediated activation of the 5`-end suggests that the proteinbulletDNA interactions occurring at the initiation of replication are important for the termination process, if this activation is maintained during replication. We envisage termination of pMV158 RC replication as the result of the following reactions: (i) introduction of a second nick at the GpA dinucleotide, the A residue belonging to the newly synthesized DNA and becoming activated (perhaps by a new RepB molecule); (ii) a closing reaction between the pG-OH end of the newly synthesized DNA and the A residue activated in this second nicking reaction, with the concomitant release of a dsDNA molecule, and (iii) a second closing reaction between the 5`-pA end, activated at the initiation stage, and the pG-OH end generated in the second nicking reaction, which leads to the release of a circular ssDNA molecule. Activation could indicate that either the protein or the nicked DNA suffered a conformational change to keep the energy to close the nick. In our opinion, the first possibility seems more likely. However, as in the case of gpII protein from filamentous coliphages, how the energy of the RepB-cleavage is stored for the ligation reaction is unknown (Kornberg and Baker, 1992). Consequently, the nicking-closing reaction mediated by RepB more closely resembles that exerted by the gpII protein of coliphage fd (Meyer and Geider, 1979) than those by Rep proteins of the RC-plasmids of the pT181 family (Thomas et al., 1990, Wang et al., 1992, Dempsey et al., 1992; Rasooly et al., 1994). Whether RepB acts through a noncovalent nucleophylic attack toward its target or through a transient covalent bond, is not yet known.

To explain how RepB acts at a distance, a complex DNA structure for this region was proposed (de la Campa et al., 1990). A DNA intrinsic curvature appears to exist within the plasmid origin, as reflected by (i) the presence of (dT-dA)(n) tracts spaced by integer helix turns (Fig. 1B), (ii) the anomalous migration of dso-containing DNA fragments in polyacrylamide gels (not shown), and (iii) the distribution of bands of hipersensitivity to DNase I, as well as the hydroxyl radical footprinting pattern of naked DNA (de la Campa et al., 1990). Cylindrical projection of the DNA helix between coordinates 400 and 600 (not shown) indicates that the RepB nick site and the center of the iterons are located in the same face of the helix. We propose that the DNA sequence between the iterons and the nic region could provide a flexible arm (flexibility, which could be increased by Mn ions), so that the binding of RepB to the iterons could appropriately place the protein close to Hairpin I. Such a highly sophisticated proteinbulletDNA interplay should lead to local unwinding of the DNA and to the exposure of the nick site in a single-stranded form. Whether this spacer acts as an enhacer for replication, as is the case of filamentous phages (Horiuchi, 1986), is not known. Be that as it may, the actual mechanism of termination of replication by RepB is intriguing. The elegant model proposed by Rasooly and Novick(1993) for termination of pT181 replication is based on the inactivation of RepC protein through generation of a RepC heterodimer. However, in the case of RepB of pMV158, the lack of stable covalent binding of RepB to DNA would indicate that this mechanism of inactivation does not occur in pMV158. Yet, the plasmid copy number is subjected to a strict control which, in addition to the role of the plasmid-encoded repressors (del Solar and Espinosa, 1992; Fig. 1), should involve lack of recycling of the initiator protein.


FOOTNOTES

*
This research was supported by Comisión Interministerial de Ciencia y Tecnología (Grants BIO94-1029 and BIO92-1018-CO2-02), by the Comunidad Autónoma de Madrid (grant 190/92), and in part by the European Communities (project CHRX-CT92-0010). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M28538[GenBank].

§
To whom correspondence should be addressed. Tel.: 34-1-5611800 (ext. 4209); Fax: 34-1-5627518; CIBME13{at}cc.csic.es.

(^1)
The abbreviations used are: RC, rolling circle; ds, double-stranded; nt, nucleotide(s); ss, single-stranded; bp, base pair(s); HPLC, high performance liquid chromatography.

(^2)
M. Moscoso, G. del Solar, and M. Espinosa, unpublished observations.

(^3)
M. Moscoso, unpublished observations.


ACKNOWLEDGEMENTS

We thank E. Lanka for discussions, fruitful advice and critical reading of the manuscript; W. Pansegrau for supplying information on the nicking reactions; J. M. Andreu and G. Rivas for conducting experiments with the analytical ultracentrifuge; J. Varela for care with the oligonucleotide syntheses; and G. Giménez and R. Eritja for advice in attempting to solve the chemistry problems. We also thank M. T. Alda, P. Valiente, and R. Galán and D. M^cIntosh Main for corrections in English.


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