Nucleic Acids Research, 2001, Vol. 29, No. 2 536-544
© 2001 Oxford University Press
Cnr interferes with dimerization of the replication protein 
in phage-plasmid P4
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Via Celoria 26, 20133 Milano, Italy
Received August 4, 2000; Revised October 20, 2000; Accepted November 17, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein
. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in (cr) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on 
immunity in E.coli for in vivo detection of protein–protein interactions, we found that: (i) protein interacts with Cnr, whereas cr proteins do not; (ii) both – and cr–cr interactions occur and the interaction domain is located within the C-terminal of ; (iii) Cnr–Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both – and cr–cr dimerization. Our data suggest that Cnr and interact in at least two ways, which may have different functional roles in P4 replication control. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P4 is a natural phasmid that can propagate in Escherichia coli both as a temperate phage and as a plasmid (1–3). The double-stranded P4 DNA circularizes after infection and replication starts from a single site, ori1, proceeding bidirectionally in a
-type mode (4). DNA replication depends on the product of the P4 gene, a multifunctional protein organized in distinct domains: the N-terminal region exhibits primase activity, the middle and C-terminal parts display helicase activity and the C-terminal end exhibits DNA binding activity (Fig. 1) (5). Two sites in the P4 genome, ori1 and crr, are essential in cis for replication. Both contain several direct and inverted repeats of a decameric sequence, the type I iterons (6,7), which are bound by the protein (8). Although essential for replication (6,7), crr is not an origin of replication (4,9). In several iteron-containing plasmids (such as P1, R6K, RK2) (10,11) the replication protein binds to specific sites and DNA looping and/or intermolecular pairing of DNA molecules, mediated by protein–protein interactions, occurs. The formation of the multimeric protein–DNA complexes (handcuffing; 12) inhibits replication initiation and allows plasmid copy number control. However in P4, unlike the above model, crr is positively required in cis for replication and does not appear to be involved in P4 DNA replication control (4,7,13).
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Regulation of P4 DNA replication is achieved at different levels. A first level depends on modulation of the expression of phage genes that code for replication functions (2,14–16). However, this regulation is not sufficient to control P4 copy number when P4 propagates as a plasmid. In this case, the P4 Cnr (copy number regulation) protein is essential to modulate the activity of
protein (13,17,18). Deletion of the P4 cnr gene causes P4 DNA over-replication and cell lethality, thus preventing P4 propagation in the plasmid state (13,17); whereas overexpression of Cnr leads to inhibition of P4 DNA replication. However, if the expression of both the Cnr and proteins is increased, no inhibition of DNA synthesis is observed (17). This suggested that the control of P4 DNA replication depends on the relative concentration of the Cnr and proteins.
P4 mutants insensitive to the Cnr control carry amino acid substitutions in the C-terminus of protein (cr mutations; 18) (Fig. 1). All such mutants are impaired in plasmid propagation. The cr mutations are in the DNA-binding domain of , which has been mapped to within a 141-amino acid region, near the C-terminus of the protein (19). Four mutations are clustered (G732V, G732W, L733V and L737V) and a fifth mutation maps at some distance (T675M). This localization suggests that the negative control of Cnr is exerted through a direct interaction with .
It has been shown in vitro that the Cnr protein increases affinity for ori1 and crr binding, whereas such an effect could not be observed on cr mutant proteins (18). It was thus hypothesized that Cnr increases the affinity of the protein for the origin of replication; however, interaction between the two proteins has not been demonstrated in vivo.
In this work, by making use of the two-hybrid system in yeast and the CI dimerization test in E.coli, we investigated in vivo interactions of and Cnr proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microorganisms and media
Manipulation of bacterial as well as yeast strains and of nucleic acids and proteins was carried out using standard methods (20,21). The E.coli K12 strains used were CSH50 [
(pro-Lac) F'(proABlacIqZM15, traD36)] (22) and 71–18 [(Lac-proAB) F'lacIq, lacZM15 pro supE] (20). The Saccharomyces cerevisiae strain was EGY48 (MAT, his3, trp1, ura3-52, leu2::LexAop6-LEU2) (21). Yeast transformation was performed by the lithium acetate procedure (23). Yeast SD medium (23), containing the specific amino acids and/or uracil, supplemented with 2% glucose or 2% galactose/1% raffinose (w/v), was used.
Bacteriophages and plasmids
The bacteriophage strains used were: wild-type, cY17 (24) and 668 (25). The plasmids are listed in Table 1. The plasmids used in the two-hybrid system are described in detail by Golemis et al. (21). The plasmids used for the immunity system are described by Castagnoli et al. (26) and Longo et al. (27).
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Identification of protein–protein interactions
Two different systems were used for the identification of protein–protein interactions. The two-hybrid system in S.cerevisiae was performed as described previously (21). Experimental details are reported in the legend to Table 2.
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The
immunity system is described by Castagnoli et al. (26) and the competition essay by Longo et al. (27).
Efficiency of plating of
Overnight cultures of CSH50, carrying the different plasmids, were grown at 37°C with aeration in TB broth, supplemented with 0.2% maltose and 0.01 M MgSO4 and ampicillin (50 µg/ml) or chloramphenicol (30 µg/ml), as required. In the case of low copy number plasmids, 1 mM isopropyl-ß-D-thiogalactoside (IPTG) was added to the medium. Cultures of both high and low copy number plasmids (0.3 ml) were plated with soft agar containing 1 mM IPTG. Drops (10 µl) of a suspension of the wild-type, the immunity sensitive cY17 and the virulent 668 mutant were streaked on the plates. The efficiency of plating relative to the control strain CSH50 was evaluated. CSH50/pC132 and CSH50/pC168, expressing the CI repressor fused to the Rop protein, were used as positive controls (26).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Detection of
–Cnr interaction by the two-hybrid system in yeastThe possibility that the Cnr protein interacts with the
protein was suggested by the existence of mutations in the P4 gene that abolish the negative control exerted by Cnr on P4 replication (cr mutations; 18) (Fig. 1). To demonstrate Cnr– interaction in vivo, we used the two-hybrid system in yeast (21): the wild-type gene and four cr mutant genes (T675M, G732V, G732W and L733V) were fused to the LexA DNA-binding domain in pEG202 (pGM585, pGM590, pGM589, pGM588 and pGM587, respectively), whereas cnr was fused to the B42 activation domain in pJG4–5 (pGM592). After transformation of the S.cerevisiae strain EGY48 (pSH18–34), –Cnr interaction was revealed by the expression of two different reporter genes: the chromosomal LEU2 gene and the plasmid lacZ gene, both under control of LexA operators. The results, reported in Table 2, showed that concomitant expression in EGY48 (pSH18–34) of Lex
wild-type and B42Cnr activated both the LEU2 and the lacZ reporter genes, indicating that the two proteins interact with each other.
When the LexAcr mutant hybrid proteins crG732V, crG732W or crL733V and B42Cnr were expressed in the same strain, activation of the reporter genes was not observed. Thus, Cnr does not interact with these cr proteins. A low level of activation of the chromosomal LEU2 gene, but not of the plasmid lacZ reporter gene, was detected with the LexAT675M protein and might indicate leakiness of the mutant.
Detection of – interactions by the two-hybrid system in yeast
Using the two-hybrid system in yeast, we also investigated whether proteins were able to self-interact. The wild-type protein was fused both to the LexA DNA-binding domain and to the B42 transactivation domain (pGM591). The results, reported in Table 3, showed that only co-expression of LexA and B42 led to activation of LEU2 and lacZ reporter genes. Thus, proteins interact with each other.
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We also tested the ability of a mutant
crG732W protein to dimerize. Both cr–cr homo- and cr– heterodimerization was observed (Table 3), indicating that the presence of the crG732W mutation did not impair – interaction.
Identification of the interaction domain
The protein is 777 amino acids long and has a modular organization (5). We first attempted to identify its interaction domain by the two-hybrid system in yeast. However, fusion of portions of the proteins with LexA produced false positives. Thus, we used an in vivo assay for detection of protein–protein interactions in E.coli, based on fusions with the CI repressor. In this system, the N-terminal part of the CI repressor, which contains the DNA-binding domain, is fused to the protein to be tested. A truncated CI protein, which lacks the C-terminal dimerization domain, is inactive. However, if the fused polypeptide can dimerize, CI functionality is restored and the hybrid protein confers immunity to infection (24,26,28).
CI fusion with the whole protein did not express immunity (data not shown); this might be due to steric hindrance of the fusion protein. Thus, fragments of decreasing length of the C-terminal part of the gene were cloned, creating fusions with the N-terminal part of CI; the different regions used are indicated in Figure 1. Strain CSH50 was transformed with the plasmids and the resistance to infection tested. Four constructs displayed immunity and they all cover the C-terminal part of the gene. The smallest fragment contains the portion from amino acid 707 to 747 (pGM730). Neither the 724–777 nor the 707–730 regions expressed immunity (pGM731 and pGM729). Thus, the results of the immunity analysis indicated that protein residues 707–747 are critical for dimerization in vivo.
Most cr mutations map in this region (Fig. 1). Thus, we tested whether CIcr fusion proteins could dimerize. The DNA regions encoding the 620–777 amino acids from crG732W, G732V and T675M fused to CI conferred immunity (Table 4), indicating that the cr mutations did not affect dimerization ability.
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Detection of Cnr–Cnr interactions in vivo
In order to test whether Cnr–Cnr interactions occurred, the Cnr protein was fused to the
CI DNA-binding domain (pGM726). Expression of the CICnr hybrid repressor conferred immunity to infection (Table 4), indicating that Cnr proteins can interact with each other.
Cnr interferes with – interactions
We have shown that the interaction domain of the protein is localized in its C-terminal part, in which most cr mutations are mapped. This suggests that the same region of the protein could be involved in both – and Cnr– interactions and that Cnr could interfere with – interaction. To test this hypothesis, we used a competition assay, based on the immunity system (27). In this assay, the CI fusion proteins were expressed from a low copy number plasmid and the competitor Cnr protein was fused to a mutant CI repressor (CI*), unable to bind DNA, and expressed in the same strain from a high copy number plasmid (pGM727). Interaction of CI*Cnr fusion protein with CI would compete with CI–CI interactions, thus preventing expression of immunity.
CI hybrid proteins carrying the 620–777, 664–777 and 741–777 regions were expressed from low copy number plasmids (pGM776, pGM738 and pGM740, respectively: the latter was used as a negative control). The hybrid CIRop protein, expressed from pC168, was used as a positive control (27). The results are reported in Table 5A. As expected, both pGM776 and pGM738 conferred immunity to the CSH50 host when induced with IPTG, whereas pGM740 did not.
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Coexpression of the CI*
Cnr protein from a high copy number plasmid (pGM727) with either CI620–777 or CI664–777 restored sensitivity, whereas coexpression of the control protein CI*Rop from a high copy number plasmid (pC169) did not alter immunity (Fig. 2; Table 5B). On the other hand, CI*Cnr neither interfered with immunity expressed from pC168 (CIRop) nor altered plating ability on CSH50/pGM740 (CI741–777).
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To test if Cnr could interfere with
cr–cr interactions, hybrid CIcr proteins, carrying the 620–777 region with either G732W, G732V or T675M mutations were cloned in the low copy number vector (pGM778, pGM779 and pGM780, respectively). Surprisingly, CI*Cnr efficiently competed with dimerization of all three CIcr fusion proteins (Table 5C). Thus, it appears that the cr mutations do not prevent interference of Cnr with cr–cr interactions.
It is possible that the above result depends on overexpression of CI*Cnr from the high copy number plasmid pGM727. Thus, we varied the concentration of Cnr relative to by making use of compatible plasmids either in low or high copy number, and we compared the effects on immunity expressed from wild-type CI620–777 and the mutant CIcr620–777G732V. We found that: (i) the wild-type Cnr protein expressed from a high copy number plasmid (pGM283) efficiently competed both CI and CIcr dimerization; (ii) expression of Cnr from the low copy number plasmid pST106 caused a comparably weak interference with immunity with either + and cr constructs; (iii) in both + and cr constructs, CI dimerization could not be competed by Cnr when CI was expressed from a high copy number plasmid (Table 5D). Thus, although Cnr interference with immunity can be modulated, a similar effect was observed with both wild-type and mutant cr proteins. Our data indicate that Cnr is able to interfere with both – and cr–cr interactions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interaction between
protein and CnrAutonomous P4 DNA replication, which occurs both in the lytic cycle and in the plasmid state, depends on the
protein. In vitro, P4 DNA replication does not require other P4-encoded proteins (9). In vivo, the Cnr protein is essential for P4 maintenance in the plasmid state. In the absence of Cnr, as in P4 cnr deletion mutants, P4 over-replicates and plasmid propagation is impaired (17). Moreover, overexpression of Cnr inhibits P4 DNA replication. Thus, the cnr gene appears to encode a negative regulator that is required for plasmid copy number control.
P4 cr mutants, isolated by their ability to grow on E.coli that overexpressed the Cnr protein (18), map in the C-terminal part of the protein. This suggests that the negative control of Cnr is exerted through a direct interaction with .
In this work, using the two-hybrid system in S.cerevisiae, we have shown that the protein can interact with the Cnr protein. However, Cnr does not interact with cr mutant proteins, with the exception of crT675M, in which a low level of activation of the LEU2 reporter gene was observed. This suggests that the P4 crT675M mutation, which maps 60 bp apart from the others, might be less relevant for the Cnr– interaction.
The above results indicate that is the target of the Cnr protein and suggest that Cnr– interaction is required for negative regulation of DNA replication. Phage-plasmid P4 is the first example in which copy number control is carried out by a protein that interacts with the replication protein and inhibits its activity.
It has been shown that, in vitro, the Cnr protein increases binding affinity to ori1 and crr (18). It may be hypothesized that Cnr interaction with modifies its structure and increases its ability to bind DNA. In fact, Ziegelin et al. (19) observed that the truncated C-terminal region has higher affinity for DNA than the complete protein, suggesting that the N-terminus quenches the DNA binding potential of the C-terminus and the interaction with other proteins may increase DNA-binding activity.
We suggest that –Cnr efficiently competes with for ori1 and crr binding sites on P4 DNA and that the –Cnr complex is not proficient for replication. It is not known which step of P4 replication is inhibited by Cnr. Preliminary in vitro results suggested that neither primase nor helicase activities are inhibited by Cnr (18); DNA unwinding, primer synthesis or replication fork progression remain potential candidates.
Dimerization of protein
Using the two-hybrid system we found that proteins can interact with each other and the presence of the crG732W mutation does not prevent formation of both homo- and heterodimers. Using fusions with the CI repressor DNA-binding domain, we could locate the dimerization domain to the 4729–4851 P4 DNA region, corresponding to residues 707–747 of . This region overlaps the cluster of cr mutations. Nevertheless, none of the cr mutations tested affected – interaction, as can be deduced by the ability to confer immunity to infection also when expressed at low concentration. Thus, it is possible that the residues changed by the cr mutations are not directly involved in – interaction. However, it should be emphasized that the P4 cr mutants were selected for their ability to replicate in the presence of high levels of Cnr. If – interactions are essential for P4 replication the selection constraints might have screened a specific subset of mutants affected in Cnr– interaction that still conserve dimerization ability.
The system used in this work to identify protein–protein interactions is based on the expression of immunity to infection. It is known that the CI repressor not only binds DNA as a dimer, but also forms tetramers and higher order oligomers by cooperative binding via its C-terminal domain (29). Thus, immunity observed with CI fusion proteins might indicate that proteins are able to oligomerize.
In vivo, – interaction might occur between both free proteins and DNA-bound proteins. In this latter case, the subunits may be bound to the same site (either ori1 or crr) or to different sites (both ori1 and crr). Looping of P4 DNA molecules between ori1 and crr sites bound to has been observed by electron microscopy (8,19,30). This suggests that proteins, bound to ori1 and crr, might interact with each other to form an ordered structure competent for replication initiation. If different molecules are required to carry out the primase and helicase activities, interaction might be required to bring the molecules to the origin of replication. Thus, – interaction might be an essential event in the process of P4 DNA replication. Making use of the dimerization assay it will be possible to isolate mutants affected in dimerization and test their replication ability.
Cnr interferes with – interaction
We observed that immunity conferred by CI–CI interactions was efficiently inhibited by the Cnr protein. Competition occurs also with CIcr mutant proteins. These data suggest that two different types of interaction are possible between and Cnr. The first, revealed by the two-hybrid test, is impaired by cr mutations. The second is highlighted by Cnr competition with – interactions in the system. This second type of interaction implies different contacts between the two proteins since, unlike the first one, it is not affected by cr mutations.
It may be hypothesized that Cnr contacts – complexes and causes a structural change of the multiprotein complex, thus modifying its functional role. The two types of interaction could be sequential: Cnr first interacts and modifies complexes, thus increasing affinity for DNA, then binds to and interferes with its replication ability. The latter event would not occur with cr mutant proteins. This observation also implies that Cnr interaction with complexes does not directly inhibit P4 DNA replication.
Alternatively, – interactions might be essential for the formation of an active replication complex, and the cr mutations are a subset of mutants that still retain this ability. Thus, a simple model where Cnr interferes with – interaction and this inhibits replication is still tenable. This model could be tested, for example, by isolating mutants in either – or –Cnr interactions by a two-hybrid system and/or dimerization assay. Such mutants could then be analyzed for their replication proficiency and the reciprocal –Cnr and – interactions.
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ACKNOWLEDGEMENTS |
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We thank R. Brent, R. Calendar, J. Eriksson and F. Gigliani for kindly providing the strains and plasmids used in this work. We are grateful to E. Boye, E. Haggård-Ljungquist and P. Plevani for helpful discussions and suggestions. This work was supported by grant no. 98.00458.CT04 of the Consiglio Nazionale delle Ricerche, Rome, Italy, and by grants from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Rome, Italy.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +39 02 26605217; Fax: +39 02 2664551; Email: daniela.ghisotti{at}unimi.it Present addresses:Arianna Tocchetti, Department of Medical Biochemistry, University of Göteborg, Medicinaregatan 9A, Box 440, 40530 Göteborg, SwedenStefania Serina, Biosearch Italia, Via R. Lepetit 34, 21040 Gerenzano (VA), Italy
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