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• W2041152090 abstract "Protein p6 of Bacillus subtilis phage Ø29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of Ø29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate Ø29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association. Protein p6 of Bacillus subtilis phage Ø29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of Ø29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate Ø29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association. base pairs(s) oligomerization domain DNA binding domain wild-type plaque-forming units polyethyleneimine a, apparent weight-average molecular weight isopropyl β-d-thiogalactopyranoside polyacrylamide gel electrophoresis DNA transactions, such as DNA replication and transcription, require higher-order DNA-protein complexes, the assembly of which is sometimes facilitated by proteins with an architectural function. This is the case of the nucleoid-associated proteins, of which the ones most extensively studied are HU, H-NS, IHF, and FIS. They all bind and bend DNA, sharing some common characteristics such as being very abundant, having a small size, and playing a pleiotropic role. The function of these proteins requires formation of dimers or oligomers. Thus, HU is a homodimer in most bacteria, although it is a heterodimer inEscherichia coli (1Rouvière-Yaniv J. Kjeldgaard N.O. FEBS Lett. 1979; 106: 297-300Crossref PubMed Scopus (102) Google Scholar, 2Claret L. Rouvière-Yaniv J. J. Mol. Biol. 1997; 273: 93-104Crossref PubMed Scopus (149) Google Scholar), IHF is a heterodimeric protein encoded by two genes, himA (3Miller H.I. Cold Spring Harbor Symp. Quant. Biol. 1984; 43: 1121-1126Crossref Google Scholar) and hip (4Flamm E.L. Weisberg R.A. J. Mol. Biol. 1985; 183: 117-128Crossref PubMed Scopus (105) Google Scholar), and FIS is a homodimer (5Johnson R.C. Ball C.A. Pfeffer D. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3484-3488Crossref PubMed Scopus (96) Google Scholar, 6Koch C. Vandekerckhove J. Kahmann R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4237-4241Crossref PubMed Scopus (92) Google Scholar). On the other hand, H-NS monomers undergo self-association to form tetramers (7Ceschini S. Lupidi G. Coletta M. Pon C.L. Fioretti E. Angeletti M. J. Biol. Chem. 2000; 275: 729-734Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The N-terminal domain of H-NS is involved in oligomerization (8Ueguchi C. Seto C. Suzuki T. Mizuno T. J. Mol. Biol. 1997; 274: 145-151Crossref PubMed Scopus (86) Google Scholar, 9Williams R.M. Rimsky S. Buc H. J. Bacteriol. 1996; 178: 4335-4343Crossref PubMed Google Scholar), and the oligomeric structure of H-NS is necessary for recognition of intrinsically curved DNA and bending (10Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (165) Google Scholar). Protein p6 of Bacillus subtilis phage Ø29 is requiredin vivo for viral genome replication (11Talavera A. Salas M. Viñuela E. Eur. J. Biochem. 1972; 31: 367-371Crossref PubMed Scopus (35) Google Scholar, 12Carrascosa J.L. Camacho A. Moreno F. Jiménez F. Mellado R.P. Viñuela E. Salas M. Eur. J. Biochem. 1976; 66: 229-241Crossref PubMed Scopus (90) Google Scholar) and repression of transcription from the early promoter C2 (13Whiteley H.R. Ramey W.D. Spiegelman G.B. Holder R.D. Virology. 1986; 155: 392-401Crossref PubMed Scopus (28) Google Scholar). In vitrostudies have shown that protein p6 is involved both in replication and transcription; it stimulates the initiation and the transition to elongation steps of Ø29 DNA replication (14Pastrana R. Lázaro J.M. Blanco L. Garcı́a J.A. Méndez E. Salas M. Nucleic Acids Res. 1985; 13: 3083-3100Crossref PubMed Scopus (61) Google Scholar, 15Blanco L. Gutiérrez J. Lázaro J.M. Bernad A. Salas M. Nucleic Acids Res. 1986; 14: 4923-4937Crossref PubMed Scopus (60) Google Scholar), represses the early C2 promoter (16Barthelemy I. Mellado R.P. Salas M. J. Virol. 1989; 63: 460-462Crossref PubMed Google Scholar), and regulates the switch between early and late transcription (17Elı́as-Arnanz M. Salas M. Genes Dev. 1999; 13: 2502-2513Crossref PubMed Scopus (30) Google Scholar). These functions are accomplished by the formation of a nucleoprotein complex in which the DNA adopts a right-handed toroidal conformation, and thus wrapping around a multimeric protein p6 core (18Serrano M. Gutiérrez C. Salas M. Hermoso J.M. J. Mol. Biol. 1993; 230: 248-259Crossref PubMed Scopus (37) Google Scholar). The number of copies of protein p6 in B. subtilis cells at late times of Ø29 infection has been calculated to be 6.6 × 105, enough to bind the entire viral progeny DNA (19Abril A.M. Salas M. Andreu J.M. Hermoso J.M. Rivas G. Biochemistry. 1997; 36: 11901-11908Crossref PubMed Scopus (38) Google Scholar). This, together with the ability to bind in vitro to the whole Ø29 genome, led us to propose a structural role in compacting and organizing the viral genome (20Gutiérrez C. Freire R. Salas M. Hermoso J.M. EMBO J. 1994; 13: 269-276Crossref PubMed Scopus (35) Google Scholar). The amounts of other histone-like proteins are cell cycle-dependent; thus, for E. coli cells in logarithmic growth, the most abundant ones are FIS and HU, with 1.2 × 104 and 1.5 × 105 copies per cell, respectively, whereas at stationary phase the most abundant is IHF with 1.2 × 104 copies per cell (21Azam T.A. Iwata A. Nishimura A. Ueda S. Ishihama A. J. Bacteriol. 1999; 181: 6361-6370Crossref PubMed Google Scholar). With these amounts, only a minor part of the bacterial genome would be bound by these proteins. Sedimentation equilibrium studies have shown that protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo (19Abril A.M. Salas M. Andreu J.M. Hermoso J.M. Rivas G. Biochemistry. 1997; 36: 11901-11908Crossref PubMed Scopus (38) Google Scholar). These oligomeric structures have been observed by transmission electron microscopy (22Abril A.M. Marco S. Carrascosa J.L. Salas M. Hermoso J.M. J. Mol. Biol. 1999; 292: 581-588Crossref PubMed Scopus (11) Google Scholar), and their structure, as deduced by image processing, is compatible with that described for the path followed by the DNA in the protein p6·DNA complex (18Serrano M. Gutiérrez C. Salas M. Hermoso J.M. J. Mol. Biol. 1993; 230: 248-259Crossref PubMed Scopus (37) Google Scholar). Thus, it has been proposed that protein p6 could act as a scaffold organizing the DNA into the appropriate configuration. Protein p6 binding to DNA is highly cooperative 1A. M. Abril, unpublished results. and extends throughout the whole Ø29 DNA forming multiple complexes of very heterogeneous sizes; the minimal size ranges from ∼130 base pairs (bp)2 observed by electron microscopy after psoralen cross-linking, to ∼80–90 bp, shown by protection of micrococcal nuclease digestion (20Gutiérrez C. Freire R. Salas M. Hermoso J.M. EMBO J. 1994; 13: 269-276Crossref PubMed Scopus (35) Google Scholar). Thus, multiple protein-DNA and protein-protein interactions are required to stabilize the complex, suggesting that association equilibria among protein p6 oligomers would modulate their interaction with DNA. In this work we have searched regions involved in protein p6 self-interaction. The characterization of deletion mutants has suggested that the N-terminal region is involved in protein p6 self-association, and the C-terminal acidic region interferes with it. We have obtained a collection of protein p6 random mutants unable to form dimers by using an in vivo self-association assay. The mutations were mainly clustered in two regions, located at the N-terminal and central parts of the protein. This allowed us to design site-directed mutants, which have been tested in vitro. The results obtained indicate that protein p6 dimer formation is drastically reduced by mutation I8T, whereas mutation A44V impairs dimerization and decreases the activation of Ø29 DNA initiation of replication. Isopropyl β-d-thiogalactopyranoside (IPTG) and ampicillin were acquired from Sigma. Restriction enzymes (HindIII, EcoRV, BamHI, andDraI), Vent polymerase, and T4-polynucleotide kinase were obtained from New England BioLabs, Taq polymerase from Perkin-Elmer, oligonucleotides from Genset Oligos, dNTP from Amersham Pharmacia Biotech, [α-32P]dATP (3000 Ci/mmol) and [γ-32P]ATP (3000 Ci/mmol) from Amersham International plc. Glutaraldehyde was purchased from Serva. The plasmid pBF21, containing the λ cI gene under the control of a tandem lacUV5 promoter-operator region (10Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (165) Google Scholar), was digested with HindIII and EcoRV to remove thecI gene fragment encoding the oligomerization domain (OD) (Fig. 1). Plasmids pΔcIp6 and pΔcIp6m were made by a polymerase chain reaction (PCR) step cloning procedure. Wild-type (wt) gene 6, cloned in plasmid pPR55w6 (23Bravo A. Hermoso J.M. Salas M. Mol. Gen. Genet. 1994; 245: 529-536Crossref PubMed Scopus (25) Google Scholar), and mutant gene 6R6A, from plasmid pPR55R6A (23Bravo A. Hermoso J.M. Salas M. Mol. Gen. Genet. 1994; 245: 529-536Crossref PubMed Scopus (25) Google Scholar), were obtained by PCR by using primers designed to introduce HindIII andEcoRV restriction sites 5′-GAAAGTGGGAAAGCTTTATGGCAA-3′ and 5′-CCTTCTCTTGTGATATCATCATTCAGC-3′, respectively. PCRs were carried out with 1 μm oligonucleotides, 0.2 μg of pPR55w6 or pPR55R6A as templates, 100 μm dNTP, and 2 units of Vent polymerase on its reaction buffer. PCR fragments were cloned into digested pBF21, to generate plasmids pΔcIp6 and pΔcIp6m, containing chimeric genes encoding for the DNA binding domain of the cI repressor, cI(DBD), fused to those encoding for p6wt protein or to mutant protein p6R6A, respectively (Fig. 1). The wt and mutant gene 6 were sequenced from the plasmids. Random mutagenesis of R6A single mutant gene 6 was performed on plasmid pΔcIp6m. The oligonucleotides used generated HindIII and EcoRV restriction sites (5′-GGCTCCAAGCCAAGCTTTATG-3′ and 5′-CGGAATGGACGATATCATCA-3′). To obtain random mutations, PCR was carried out under two different conditions: one had limited dATP (20 μm) and the other had additionally 0.5 mm MnCl2 (24Arcangioli B. Ghazvini M. Ribes V. Nucleic Acids Res. 1994; 22: 2930-2937Crossref PubMed Scopus (9) Google Scholar, 25Leung D.W. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar). Thus, the reactions contained 1 μm oligonucleotides; 0.2 μg of plasmid; 1.25 mm MgCl2; 5 units of Taqpolymerase; 100 μm each of dCTP, dGTP, and dTTP; and 20 μm dATP, in the absence or in the presence of 0.5 mm MnCl2 in Taq polymerase buffer. The mutated fragments were digested with HindIII andEcoRV and cloned into plasmid pBF21 digested with the same enzymes. E. coli 71-18 lacIq cells (26Yanisch-Perron C. Vieira J. Messing J. Gene. 1985; 33: 103-119Crossref PubMed Scopus (11471) Google Scholar) were transformed with these constructions and tested for λ phage development as described below. The mutated gene 6 was sequenced in those clones showing a lytic phenotype. E. coli 71-18lacIq cells transformed with plasmid pBF21 or the derivatives described above, were grown at 37 °C up to an optical density of 0.6 at 620 nm in LB medium containing ampicillin (100 μg/ml). The production of chimeric proteins was induced by addition of 250 μl of 100 mm IPTG to 0.4 ml of culture. After induction, cells were infected with λ146 hypervirulent phage (27Bailone A. Devoret R. Virology. 1978; 84: 547-550Crossref PubMed Scopus (24) Google Scholar), essentially as described (10Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (165) Google Scholar). After 5-min incubation, 5 ml of LB medium containing ampicillin (100 μg/ml) was added to the infected cultures, which were further incubated at 37 °C. At the indicated times, 0.4-ml aliquots were centrifuged and 150 μl of supernatant was mixed with 30 μl of chloroform. The number of plaques forming units per milliliter in each supernatant was determined using E. coli 71-18 lacIq as the recipient strain. Site-directed mutants of wt gene 6 were constructed by PCR using plasmid pPR55w6 (23Bravo A. Hermoso J.M. Salas M. Mol. Gen. Genet. 1994; 245: 529-536Crossref PubMed Scopus (25) Google Scholar) as template. As primers, we have used four oligonucleotides with degenerated positions, to obtain mutations in amino acids 7, 8, 43, and 44 of protein p6 (5′-CGGTTGTCTTTGTGRTTWCTCTCTGC-3′; 5′-GCAGAGAGWAAYCACAAAGACAACCG-3′; 5′-CTCCATTGARCTYGTTCCATTGTC-3′; 5′-GACAATGGAACRAGYTCAATGGAG-3′). Two oligonucleotides 5′-CGACCTGCAGGGATCCGGCC-3′ and 5′-CTAATACTAGAAGCTTCTCTTGTG-3′ were used to generateBamHI and HindIII restriction sites to clone the fragments containing mutated gene 6 into plasmid pPLc28 (28Remaut E. Stanssens P. Fiers W. Gene. 1981; 15: 81-93Crossref PubMed Scopus (466) Google Scholar) under the control of λ PL promoter. The λ lysogen E. coli K-12ΔH1Δtrp encoding the thermosensitive cI857 repressor (28Remaut E. Stanssens P. Fiers W. Gene. 1981; 15: 81-93Crossref PubMed Scopus (466) Google Scholar) was electroporated with the recombinant plasmids to obtain a library of random mutants. The mutations were determined by sequencing the corresponding gene 6 in selected clones from the library. Cultures ofE. coli K-12ΔH1Δtrp cells transformed with the recombinant plasmids pMJnΔ5 and pMJnΔ13 (29Otero M.J. Lázaro J.M. Salas M. Gene. 1990; 95: 25-30Crossref PubMed Scopus (9) Google Scholar) and pMJcΔ14 and pMJcΔ16 (30Otero M.J. Salas M. Nucleic Acids Res. 1989; 17: 4567-4577Crossref PubMed Scopus (7) Google Scholar) were induced for 1 h at 42 °C. After centrifugation, 20 g of cells was ground with alumina and extracted with buffer A (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 7 mm β-mercaptoethanol, 5% glycerol) with 0.5 m NaCl. Alumina and cell debris were removed by centrifugation. Polyethyleneimine (PEI) was added to the supernatant up to 0.25%, after adjusting absorbance at 260 nm to 120 units/ml, and centrifuged for 10 min at 10,000 × g to remove DNA. The supernatant was made 0.35 m NaCl with buffer A; deletion mutant proteins were recovered in the pellet after centrifugation as above. To eliminate PEI, pellets were resuspended in buffer A containing 0.4 m NaCl, and the proteins were precipitated with ammonium sulfate up to 70% saturation. Pellets containing N-terminal deletion mutants, p6NΔ5 and p6NΔ13, were resuspended in buffer A and applied to a DEAE-cellulose column, whereas those containing C-terminal deletion mutants, p6CΔ14 and p6CΔ16, were applied to a phosphocellulose column. They were eluted with salt concentrations ranging from 75 to 200 mm NaCl. The mutant proteins were further subjected to heparin-agarose chromatography and eluted at salt concentrations from 50 to 350 mm NaCl. Minor contaminants were eliminated using Centricon-50 from Amicon; after this step the purity of the mutant proteins was estimated to be 95% by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Blue staining. Cultures of E. coli K-12ΔH1Δtrp cells, transformed with plasmids encoding protein p6 site-directed mutants, were induced for 2 h at 42 °C. After centrifugation, 6–8 g of cells was ground with alumina, and the DNA was removed as described above. The mutant proteins were extracted from the PEI pellet by washing with 1m NaCl buffer A and precipitated with ammonium sulfate to up to 70% saturation. Pellets containing mutant proteins were resuspended in buffer A and applied to a phosphocellulose column, except p6I8T, which was applied to a DEAE-cellulose column. The different protein p6 mutants were eluted with buffer A containing NaCl concentrations from 75 to 200 mm. Minor contaminants were eliminated by 15–30% glycerol gradient centrifugation. After this step all protein p6 single mutants were more than 90% homogeneous as estimated by SDS-PAGE followed by Coomassie Blue staining. The reaction mixture contained (in 25 μl) 50 mm Tris-HCl, pH 7.5; 10 mm MgCl2; 20 mm ammonium sulfate; 1 mm dithiothreitol; 10% glycerol; 0.25 μm[α-32P]dATP; 0.3 μg of phage Ø29 terminal protein-DNA complex, purified as described (31Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5522-5526Crossref PubMed Scopus (116) Google Scholar); 20 ng of terminal protein; 20 ng of Ø29 DNA polymerase; and the indicated amounts of either protein p6wt or protein p6 mutants. After incubation for 15 min at 15 °C, the reaction was stopped by adding up to 10 mmEDTA and 0.1% SDS. The samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS. The initiation complex formed was analyzed by SDS-PAGE as described (31Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5522-5526Crossref PubMed Scopus (116) Google Scholar). Quantification was performed by using a Fuji BAS-1500 image analyzer. Glutaraldehyde cross-linking was carried out essentially as described (32Balzer D. Ziegelin G. Pansegrau W. Kruft V. Lanka E. Nucleic Acids Res. 1992; 20: 1851-1858Crossref PubMed Scopus (125) Google Scholar). Either wt or mutant p6 proteins, 5 μm each, were incubated at room temperature in 20 mm triethanolamine, pH 8.0, 50 mm NaCl, and 300 μm glutaraldehyde. After 45-min incubation, the reaction was stopped by adding Tris-HCl, pH 7.5, up to 150 mm. Samples were analyzed by SDS-Tricine-PAGE (33Schagger H. Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10494) Google Scholar). The experiments were performed in a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics, using an An60Ti rotor. Protein p6 was equilibrated in 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, and 50 mm NaCl. Protein p6 (100 μl of 100 μm) was centrifuged at 25,000 rpm, and absorbance scans at 280 nm were taken at sedimentation equilibrium. The equilibrium temperature was 4 °C. High speed sedimentation (42,000 rpm) was conducted afterward for baseline correction. Whole cell apparent weight average molecular weights (Mw,a ) were determined by fitting a sedimentation equilibrium model for a single sedimenting solute to individual datasets with the program EQASSOC (supplied by Beckman, Ref.34Minton A.P. Shuster T.M. Laue T.M. Modern Analytical Ultracentrifugation. Birkhauser, Boston, MA1994: 81-93Crossref Google Scholar). The partial specific volume of protein p6 was 0.728 ml/g, calculated from the amino acid composition of the protein deduced from the gene 6 sequence (35Murray C.L. Rabinowitz J.C. J. Biol. Chem. 1982; 257: 1053-1062Abstract Full Text PDF PubMed Google Scholar). DNase I footprinting was carried out essentially as described (36Prieto I. Serrano M. Lázaro J.M. Salas M. Hermoso J.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 314-318Crossref PubMed Scopus (39) Google Scholar). The indicated amounts of either wt or mutant p6 proteins were incubated with 2 ng of Ø29 DNA left terminal fragment, 259 bp long. The DNA fragment was obtained by PCR in a reaction containing 0.2 μg of Ø29 DNA as template, 1 μm oligonucleotides 5′-GCGCTTTAAAGTAAGCCCCCACCCTC-3′ and 5′-GCCCACATACTTTGTTGATTGG-3′, 100 μm dNTP, and 2 units of Vent polymerase in its reaction buffer. The last oligonucleotide was previously 5′-labeled with [γ-32P]ATP using T4 polynucleotide kinase to obtain the Ø29 DNA terminal fragment internally labeled. The PCR product was digested with DraI to restore the Ø29 DNA terminal end. DNase I digestion was carried out for 5 min at 15 °C in 25 μl of a solution containing 10 mm MgCl2. The reaction was stopped by addition of EDTA, pH 8.0, up to 20 mm final concentration. DNA was precipitated with ethanol in the presence of RNA carrier (0.25 mg/ml) and then subjected to denaturing electrophoresis in 6% (w/v) acrylamide gel. Circular dichroism spectra were recorded using a Jasco J-720 spectropolarimeter using a 0.2-cm path length cuvette. Proteins were used at room temperature at a concentration of 0.13 g/l in 10 mm Tris-HCl, pH 7.5. The final spectrum in each case is the mean of 10 measurements with a step size of 0.2 nm recorded from 260 to 200 nm. Data were base line-corrected by substraction of that of the Tris buffer. It has been reported that N-terminal deletion mutants of protein p6 lacking 5 or 13 amino acids (p6NΔ5 and p6NΔ13, respectively) were not able to interact with a Ø29 DNA terminal fragment as detected by DNase I footprinting (29Otero M.J. Lázaro J.M. Salas M. Gene. 1990; 95: 25-30Crossref PubMed Scopus (9) Google Scholar). Because DNA binding is highly cooperative1 and requires the formation of large oligomers, the lack of DNA binding could be due to a deficiency in protein p6 self-association; therefore, we tested by glutaraldehyde cross-linking the capability of both deletion mutants to self-associate. Fig.2 shows that dimer formation was highly impaired in the p6NΔ5 mutant, being about 10% that of the p6wt protein. In addition, dimers were not detected with the p6NΔ13 mutant (not shown). On the other hand, a 14-amino acid C-terminal deletion mutant of protein p6 (p6CΔ14) bound DNA 2-fold better than the p6wt protein (30Otero M.J. Salas M. Nucleic Acids Res. 1989; 17: 4567-4577Crossref PubMed Scopus (7) Google Scholar). Two C-terminal deletion mutants of 14 and 16 amino acids (p6CΔ14 and p6CΔ16, respectively) were tested for dimer formation by glutaraldehyde cross-linking. As Fig. 2 shows, the ability of dimer formation in the p6CΔ16 mutant protein was about 2-fold that of the p6wt protein; a similar result was obtained with the mutant protein p6CΔ14 (not shown). Therefore, the observed DNA binding properties of p6NΔ5, p6NΔ13, p6CΔ14, and p6CΔ16 mutants could be explained by their self-association capacities. These results indicate that the N-terminal region of protein p6 is required for glutaraldehyde cross-linking, suggesting that this region is involved in dimer formation; however, glutaraldehyde cross-linking is favored when the C-terminal region is removed, suggesting that this region impairs dimer formation. To further investigate the protein p6 regions involved in self-association, we carried out anin vivo assay in which we used the region encoding the N-terminal domain of λ cI repressor as a reporter gene for dimerization (10Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (165) Google Scholar). Phage λ cI repressor is a two-domain protein that binds DNA as a dimer; the N-terminal part is the DNA binding domain, cI(DBD), whereas the C-terminal one is the oligomerization domain, cI(OD), which is required for an efficient binding to the operator. Thus, E. coli cells expressing only cI(DBD) (ΔcI) were sensitive to hypervirulent phage λ146 infection, whereas those with intact cI were immune. Replacement of the cI(OD) by another protein provides a self-interaction assay for that protein. We have used the plasmid pBF21 (10Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (165) Google Scholar), expressing the λ phage cI repressor under the control of the lac promoter, to construct pΔcIp6 plasmid encoding a fusion protein containing the ΔcI and the protein p6wt (Fig. 1). The dimerization capacity of protein p6 was assayed by determining the development of infecting λ146 phage in E. coli cells harboring the pΔcIp6 plasmid upon induction with IPTG. Cells expressing the ΔcIp6 fusion protein were not immune to λ146 infection (Fig.3), behaving as the control cells expressing ΔcI. This result was unexpected, because protein p6 self-interacts in vitro. The expression of ΔcIp6 fusion protein was confirmed by Western blot analysis with protein p6 antiserum (not shown). An explanation for the lytic phenotype could be that the non sequence-specific DNA binding ability of protein p6 (37Serrano M. Gutiérrez J. Prieto I. Hermoso J.M. Salas M. EMBO J. 1989; 8: 1879-1885Crossref PubMed Scopus (48) Google Scholar) prevented the binding of the ΔcI to the operators. To avoid this, plasmid pΔcIp6m (see Fig. 1) was constructed, expressing a fusion containing the ΔcI and the protein p6 mutant defective in DNA binding p6R6A (38Freire R. Salas M. Hermoso J.M. EMBO J. 1994; 13: 4353-4360Crossref PubMed Scopus (22) Google Scholar). Cells harboring the plasmid pΔcIp6m showed, upon induction, a time course development of λ146 phage similar to that of the control cells expressing intact λ cI repressor. In these cases, the number of pfu/ml was about 104-fold lower than that obtained in cells expressing ΔcI (Fig. 3). Therefore, we can conclude that the mutant protein p6R6A functionally replaces the cI(OD) allowing dimerization of the ΔcI. This strongly suggests that protein p6 self-associates in vivo. The in vivoassay described above sets up the basis to select mutants in self-association. Because ΔcIp6m fusion protein confers immunity to λ146 infection, random mutagenesis was performed on mutant gene 6R6A contained in plasmid pΔcIp6m. The PCR products were cloned as above, under the control of the lac promoter, in-frame to the cI(DBD). Therefore, chimeric proteins consisting of ΔcI and random mutants of protein p6R6A were expressed. Individual colonies were tested for λ phage development as above, and clones showing a lytic phenotype were selected as candidates defective in protein p6 self-interaction. From 200 individual clones, 35 showed a lytic phenotype. Fig. 4 shows the development of λ146 phage in selected lytic clones, which was about 104-fold higher than that of cells expressing ΔcIp6m. The expression of the fusion proteins was assessed by Western immunoblotting analysis using antiserum against protein p6 (not shown). The entire nucleotide sequence of the mutated gene 6 was determined from the 35 lytic clones. Four of them had a premature stop codon. Most of the mutants had more than three amino acid changes, and only five carried double or triple mutations as shown in Fig.5. Amino acid sequence comparison with the p6R6A protein (p6m) showed that the mutations were mainly clustered in two regions: N-terminal, around position 8 (region I, Fig. 5), like those in p6m8 and p6m22 mutant proteins, and a central region around position 44 (region II, Fig. 5), like p6m23 and p6m41 mutant proteins. Mutant protein p6m94 carried a single mutation in each region. The non-random distribution of in vivo selected mutants strongly suggested the involvement of regions I and II in protein p6 self-interaction. To assess directly the involvement of individual residues in protein-protein interaction, we designed site-directed mutations on positions 7, 8, 43, and 44, where wt residues were replaced by those found in mutants selected in vivo (Fig. 5), namely E7V, I8T, Q43R, and A44V. The site-directed mutagenesis was performed by PCR on plasmid pPR55w6, encoding wt gene 6 (23Bravo A. Hermoso J.M. Salas M. Mol. Gen. Genet. 1994; 245: 529-536Crossref PubMed Scopus (25) Google Scholar). The mutated genes were cloned into an expression vector under the control of the bacteriophage λ PL promoter. Mutants were overproduced and purified up to at least 90% homogeneity. Protein p6 mutants were tested for activation of initiation of phage Ø29 DNA replication in an in vitro assay with purified proteins (14Pastrana R. Lázaro J.M. Blanco L. Garcı́a J.A. Méndez E. Salas M. Nucleic Acids Res. 1985; 13: 3083-3100Crossref PubMed Scopus (61) Google Scholar). Fig. 6 shows the results obtained with the mutant proteins p6E7V, p6I8T, p6Q43R, and p6A44V. Although the mutants p6E7V and p6Q43R showed an activation similar to that of the p6wt protein, the p6A44V mutant protein exhibited about a 6-fold reduced capacity to activate the initiation reaction. No activation was detected by the p6I8T mutant. The deficiency observed in the activation of the initiation reaction in p6A44V and p6I8T mutant proteins could be due to an impaired self-association and/or DNA binding. Because we have selected these mutants in an in vivo self-association assay, we first studied the dimerization properties of the protein p6 mutants. The effect of the p6A44V and p6I8T mutations in self-interaction was tested, as a first approach, by glutaraldehyde cross-linking. The amount of dimers formed by the p6A44V and p6I8T mutant proteins was 50% and 7%, respectively, of that found in the p6wt protein (Fig.7).Figure 7Dimer formation of protein p6wt and site-directed mutants. Glutaraldehyde cross-linking of protein p6wt and single mutants p6A44V and p6I8T. Proteins were cross-linked as described under “Experimental Procedures” and run in SDS-Tricine-PAGE, along with untreated samples. The mobilities of molecular mass standards are indicated.View Large Image Figure ViewerDownload (PPT) These results prompted us to further study the self-interaction capability of the protein p6 mutants by analytical ultracentrifugation. Sedimentation equilibrium studies had shown that protein p6, in the 1–100 μm range, is in a monomer-dimer equilibrium with a dimerization constant (K 2) of 2 × 105m−1 that shifts to higher association states at higher protein concentration (19Abril A.M. Salas M. Andreu J.M. Hermoso J.M. Rivas G. Biochemistry. 1997; 36: 11901-11908Crossref PubMed Scopus (38) Google Scholar). Fig.8 shows that, at 100 μm and 4 °C, the Mw,a ) of protein p6wt was 25,400, whereas those of p6A44V and p6I8T mutant proteins were 20,600 and 15,500, respectively, the latter being close to the theoretical value for the wt protein p6 monomer (11,900). Assuming that mutant proteins have the same self-association behavior as p6wt (19Abril A.M. Salas M. Andreu J.M. Hermoso J.M. Rivas G. Biochemistry. 1997; 36: 11901-11908Crossref PubMed Scopus (38) Google Scholar), the dimerization constant of p6A44V is slightly lower than that of the p6wt 3G. Rivas, personal communication. ; however, theK 2 of p6I8T is, at least, 10-fold lower than that of the p6wt protein.3 Therefore, we can conclude that A44V mutation slightly affects protein dimerization, which is almost abolished by the mutation I8T. Protein p6 forms a nucleoprotein complex with phage Ø29 DNA terminal fragments, in which the DNA wraps around a multimeric protein core (39Serrano M. Salas M. Hermoso J.M. Science. 1990; 248: 1012-1016Crossref PubMed Scopus (64) Google Scholar). The formation of the complex can be detected by a characteristic DNase I footprint pattern, in which strong hypersensitivities, with a periodicity of 24 bp, are located in between protected regions (36Prieto I. Serrano M. Lázaro J.M. Salas M. Hermoso J.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 314-318Crossref PubMed Scopus (39) Google Scholar). As shown in Fig. 9, protein p6A44V required a 2.5-fold higher protein amount (0.5 μg) than protein p6wt (0.2 μg) to form the complex; this may reflect the reduced capacity to form dimers of p6A44V mutant. In addition, we can observe that the digestion pattern formed with p6A44V is slightly different than that observed with p6wt. As it could be expected from the previous results, p6I8T mutant protein failed to bind DNA even at a 20-fold higher protein concentration than that of the p6wt (Fig. 9). Protein p6 of B. subtilis phage Ø29 binds DNA forming a multimeric nucleoprotein complex (39Serrano M. Salas M. Hermoso J.M. Science. 1990; 248: 1012-1016Crossref PubMed Scopus (64) Google Scholar) that is required for activation of Ø29 DNA initiation of replication (37Serrano M. Gutiérrez J. Prieto I. Hermoso J.M. Salas M. EMBO J. 1989; 8: 1879-1885Crossref PubMed Scopus (48) Google Scholar, 38Freire R. Salas M. Hermoso J.M. EMBO J. 1994; 13: 4353-4360Crossref PubMed Scopus (22) Google Scholar), repression of transcription from early C2 promoter (13Whiteley H.R. Ramey W.D. Spiegelman G.B. Holder R.D. Virology. 1986; 155: 392-401Crossref PubMed Scopus (28) Google Scholar, 16Barthelemy I. Mellado R.P. Salas M. J. Virol. 1989; 63: 460-462Crossref PubMed Google Scholar), and regulation of viral switch between early and late transcription (17Elı́as-Arnanz M. Salas M. Genes Dev. 1999; 13: 2502-2513Crossref PubMed Scopus (30) Google Scholar). Protein p6 in solution forms elongated oligomers from preformed dimers, at thein vivo estimated protein concentration (19Abril A.M. Salas M. Andreu J.M. Hermoso J.M. Rivas G. Biochemistry. 1997; 36: 11901-11908Crossref PubMed Scopus (38) Google Scholar). Oligomeric structures have been proposed to provide the scaffold on which the DNA folds into the appropriate configuration. Protein p6 binding to DNA requires multiple protein-DNA and protein-protein interactions. Thus, failure to bind DNA of N-terminal deletion mutants of 5 or 13 amino acids in protein p6 (29Otero M.J. Lázaro J.M. Salas M. Gene. 1990; 95: 25-30Crossref PubMed Scopus (9) Google Scholar), can be explained by their impaired or lost capacity, respectively, in dimer formation. We have looked for other regions involved in protein p6 self-association by using an in vivo system based on the immunity of cells expressing λ cI repressor to λ phage infection. If the λ cI(OD) is removed, immunity is lost and the infecting phage can develop lytic cycle. Thus, replacement of the cI(OD) by other protein provides a self-interaction test for this protein. When ΔcI was fused to the gene encoding p6R6A mutant protein, immunity was achieved, suggesting that the protein self-interacts in vivo. Thus, random mutants of protein p6R6A, obtained by degenerated PCR, were tested for self-association. Although the proteins contained multiple mutations, they were not randomly arranged, but mainly clustered in two regions located at the N-terminal and the central region of protein p6 (regions I and II in Fig. 5). Therefore, single site-directed mutants of protein p6wt corresponding to some of those found in vivo were constructed and assayed in vitro. Taking into account that the in vivo selected mutant proteins p6m23 and p6m41 (Fig.5) share the Q43R mutation, we would expect an involvement of Gln43 in protein p6 self-interaction. However, this was not confirmed by in vitro experiments with the corresponding site-directed mutant. The mutant protein p6Q43R did not show any significant difference with the wt protein in the activation of Ø29 DNA initiation of replication or glutaraldehyde cross-linking. A possible explanation for this result could be that the in vivo assay is more restrictive. In addition, the single mutation may not be enough to show an impaired dimerization, and additional changes might be required, like those found in the in vivoselected mutants. The involvement of region II in dimer formation is shown by p6A44V mutant protein, which has a slightly reduced capacity to form dimers. In agreement with this, DNase I footprinting (Fig. 9) shows that p6A44V has a DNA binding affinity lower than that of the p6wt protein and an impaired activation of Ø29 DNA initiation of replication. The involvement in dimer formation of region I (see Fig. 5) was expected from the results obtained with protein p6 N-terminal deletion mutants. The p6I8T mutant protein failed to activate the Ø29 DNA initiation of replication, DNA binding was not detected, and dimer formation was reduced at least 10-fold. Mutant p6 proteins are folded, as shown in Fig. 10, where the circular dichroism spectra of the wt and mutant p6 proteins do not show significant differences in their secondary structure. Altogether, these results suggest that Ile8 is involved in the self-interaction of protein p6. It has been reported that a C-terminal, 14-amino acid truncated p6 protein has a DNA binding activity 2-fold higher than that of the p6wt protein (30Otero M.J. Salas M. Nucleic Acids Res. 1989; 17: 4567-4577Crossref PubMed Scopus (7) Google Scholar). We have now shown that this truncated protein has increased its capacity to form dimers. Protein p6 has a very acidic C terminus, where 10 out of 19 residues are either Asp or Glu (see Fig.5). Therefore, it seems likely that the C-terminal acidic region modulates protein p6 self-association, interacting with a basic region. There is a cluster of basic residues overlapping region II (see Fig.5), although interaction with region I, which also contains basic residues, cannot be ruled out. This interaction could contribute to the dynamic nature of the protein p6 binding to DNA (20Gutiérrez C. Freire R. Salas M. Hermoso J.M. EMBO J. 1994; 13: 269-276Crossref PubMed Scopus (35) Google Scholar). In summary, we have found two regions involved in vivo in self-association of phage Ø29 protein p6, the N-terminal and a central region. Site-directed mutation A44V, located at the central region, impairs the protein p6 self-association. The N-terminal mutation I8T has a more drastic effect and completely abolished dimer formation. We are very grateful to Drs. R. Spurio and C. O. Gualerzi for kindly providing us plasmids pBF21 and pBF22, λ146 phage, and E. coli strain 71-18lacIq; J. Fernández and Dr. G. Rivas for their help with the analytical ultracentrifugation experiments; Dr. R. Giraldo for help in the circular dichroism experiments; J. M. Lázaro and L. Villar for the purification of wt and site-directed p6 mutant proteins; and Dr. A. Bravo for helpful discussions." @default.
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• W2041152090 title "Identification of Residues within Two Regions Involved in Self-association of Viral Histone-like Protein p6 from Phage Ø29" @default.
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• W2041152090 cites W2003453431 @default.
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• W2041152090 cites W2070851898 @default.
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