Multiple DNA Binding Activities of the Novel Site-specific Recombinase, Piv, from Moraxella lacunata
1999; Elsevier BV; Volume: 274; Issue: 14 Linguagem: Inglês
10.1074/jbc.274.14.9698
ISSN1083-351X
AutoresDeborah M. Tobiason, Anne G. Lenich, A C Glasgow,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment inMoraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the λ Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region ofM. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome. The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment inMoraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the λ Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region ofM. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome. isopropyl-1-β-d-galactopyranoside polymerase chain reaction base pair(s) kilkobase pair(s) maltose-binding protein polyacrylamide gel electrophoresis electrophoretic mobility shift assay Numerous site-specific DNA recombination systems and DNA transposition systems have been characterized biochemically and have been found to follow two distinct chemical pathways for DNA cleavage and strand transfer in recombination (reviewed in Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar, 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar, 3Landy A. Curr. Opin. Genet. Dev. 1993; 3: 699-707Crossref PubMed Scopus (85) Google Scholar, 4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). Site-specific recombination, mediated by the recombinases of the λ-integrase and Hin/resolvase families, involves a two-step transesterification reaction in which the intermediate is a covalent recombinase-DNA linkage. This covalent attachment is the result of nucleophilic attack on the DNA phosphodiester backbone by a hydroxyl group of the conserved serine (Hin/resolvase), or tyrosine (λ-integrase), of the recombinase. In the second transesterification reaction, the phosphodiester linkages of the exchanged DNA strands are restored (reviewed in Refs. 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar and 3Landy A. Curr. Opin. Genet. Dev. 1993; 3: 699-707Crossref PubMed Scopus (85) Google Scholar). In contrast, DNA transposition, mediated by transposases containing the catalytic DDE amino acid motif, utilizes a hydrolysis reaction for cleavage at the ends of the transposable element. This first cleavage leaves 3′-OH ends to act directly as the attacking nucleophile in a one-step trans-esterification reaction resulting in strand exchange. Resolution of the transposition process involves DNA replication or DNA repair activity to fill in gaps left at the target site due to the staggered cut mediated by the transposase and the 3′ hydroxyl groups at the element ends (reviewed in Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar and 4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). These features of the recombination reactions mediated by site-specific recombinases and transposases suggest that a group of related recombinases would not mediate both site-specific recombination and transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DNA inversion inMoraxella lacunata and Moraxella bovis, exhibits significant homology to the transposases of the IS110/IS492 family of IS elements (approximately 25–35% amino acid identity and 45–55% similarity, 5). Furthermore, Piv and the IS110/IS492 transposases do not appear to be related to the site-specific recombinases of the λ-Int or Hin/resolvase families or the transposases containing the DDE motif (4Mahillon J. Chandler M. Microbiol. Mol. Biol. Rev. 1998; 62: 725-774Crossref PubMed Google Scholar). Therefore, Piv and the IS110/IS492transposases may define a new family of DNA recombinases. The homology between Piv and the IS110/IS492transposases includes several highly conserved amino acid regions (5Lenich A.G. Glasgow A.C. J. Bacteriol. 1994; 176: 4160-4164Crossref PubMed Google Scholar), which, based on mutational analyses, contain functionally relevant amino acid motifs. 1D. Tobiason, A. Lenich, and A. C. Glasgow, submitted for publication. Although there is no completely conserved DNA sequence among all the IS elements and the Piv invertible DNA segment, there is a consensus sequence for the ends of a subgroup of the IS elements, which is also found overlapping the recombination sites of the Piv invertible element.1 To determine if indeed Piv and the IS110/IS492 transposases define a new family of DNA recombinases that utilize a common mechanism for both site-specific recombination and transposition, we must characterize the recombination reactions mediated by Piv and transposases from the IS110/IS492 family. As a first step in understanding the mechanism for Piv-mediated assembly of a synaptic nucleoprotein complex, DNA cleavage, and strand exchange, we have characterized the interactions of Piv with the invertible DNA segment of Moraxella lacunata. DNase I was obtained from Worthington Biochemical Corp. Amylose resin, restriction enzymes, T4 DNA ligase, Klenow (exo−), and T4 polynucleotide kinase were purchased from New England Biolabs (NEB). Pfu DNA polymerase was obtained from Stratagene. The His tag XPRESS purification system and the TA cloning kit were purchased from Invitrogen. Radionucleotides were obtained from NEN Life Science Products. Isopropyl-1-β-d-galactopyranoside (IPTG),2 dithiothreitol, and EDTA were purchased from Sigma. Dimethyl sulfate was purchased from Aldrich. The pAG607 plasmid is composed of the pMal-C2 vector (NEB) containing the piv gene inserted into theXmnI and BamHI sites such that it is in frame directly downstream of a factor Xa protease site in themalE gene. The piv gene was obtained by the polymerase chain reaction (PCR) from the pMxL1 plasmid, subclone ofM. lacunata inversion region provided by C. Marrs (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar), usingPfu DNA polymerase and the primers: 5′-GCCAGCACGTGTCTAAAACTTACATTG-3′ and 5′-CCTAAGCTTCTAGGATACCAATAAAT-3′. Similarly, the piv gene from pMxL1 was PCR-amplified with the primers: 5′-CTCGTCTCGAGTTCATGAATGCGTTTGTCAAAAG-3′ and 5′-CAGTTCACATATGTCTAAAACTTACATTGGGATT-3′, cleaved withXhoI and NdeI, and inserted into the same restriction sites in pET21a (Novagen). This placed piv in frame with a COOH-terminal His6 tag sequence, creating the Piv-His6 expression vector, pAG1300. A 245-bp fragment was generated from the pMxL1-dl24 plasmid (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar) usingPfu DNA polymerase and the primers 5′-CATAGGATCCAAAATTACCTGCCAGACATC-3′ and 5′-CCGGAATTCGCTAACCTTACACTCATAC-3′. This 245-bp fragment, containing the strong upstream binding site (sub1), was cloned into the pCR2.1 vector (TA cloning kit, Invitrogen). The resulting plasmids, pAG604 and pAG605, contain sub1 in opposite orientations. The inserted DNA was sequenced (T7 Sequenase version 2.0 DNA sequencing kit; Amersham Pharmacia Biotech) to check for any mutations due to the PCR reaction. To construct the DNA inversion test plasmid pAG862, a 5865-bp DNA fragment containing the invertible segment was obtained by partial digestion of pMxL1 with EcoRI. This fragment was gel-purified and ligated into EcoRI-digested pACYC184 (NEB) creating the pAG850 plasmid. To inactivate piv, the Ω fragment encoding the SpcR/SmR genes was recombined into the piv gene of pAG850 by homologous recombination with piv::Ω on the pMxL5 plasmid (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar) in the Rec+ strain JM101 (NEB). The recombinant plasmid, pAG862, was confirmed by restriction digestion and DNA sequencing. To produce the Piv protein fused at its amino terminus to maltose-binding protein (MBP-Piv), DH5α containing pAG607 was grown in Luria broth to OD600 = 0.5, induced with 0.5 mm IPTG, aerated at 37 °C for 2 h, and lysed in a French pressure cell. Crude extract was immediately loaded onto an amylose column, washed with column buffer (20 mm Tris-Cl, pH 7.4, 500 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol), and then eluted with 10 mm maltose in column buffer (7di Guan C. Li P. Riggs P.D. Inouye H. Gene (Amst.). 1988; 67: 21-30Crossref PubMed Scopus (546) Google Scholar, 8Maina C.V. Riggs P.D. Grandea A.G.d. Slatko B.E. Moran L.S. Tagliamonte J.A. McReynolds L.A. Guan C.D. Gene (Amst.). 1988; 74: 365-373Crossref PubMed Scopus (462) Google Scholar). Fractions were collected and frozen in 20% or 30% glycerol at −20 °C. Protein concentration was quantitated using the Bio-Rad DC protein assay kit. To produce the His6-tagged Piv, pAG1300 was introduced into BL21(DE3) (Novagen), grown to mid-log phase in Luria broth, then induced with 1 mm IPTG. After 1 h aeration at 37 °C, cells were collected by centrifugation, resuspended in 20 mm phosphate, 500 mm NaCl, pH 7.8, and lysed in a French pressure cell at 4 °C. The lysate was centrifuged at 30,000 × g. The supernatant was loaded onto an Invitrogen ProBond column (Ni2+ affinity column). Following washes with 500 mm NaCl, 20 mm phosphate, pH 7.8 buffer, protein was eluted with an imidazole step gradient (50, 200, 350, and 500 mm). Piv-His6 eluted at 300 mm imidazole. The protein was dialyzed into 20 mm phosphate, 500 mm NaCl, pH 7.8, and 30% glycerol at 4 °C and stored at −20 °C. Antibody to the Piv protein was produced in rabbits injected with purified MBP-Piv protein (BAbCO; Berkeley Antibody Company). Nonspecific antibodies were removed from the rabbit sera by absorption to whole cells of DH5α cultures, followed by multiple passages over a Sepharose column bound withEscherichia coli proteins and MBP. This affinity column was made by lysing a culture of pMal-C2/DH5α that had been induced with 0.5 mm IPTG to express the MBP protein, then binding the proteins from this lysate to cyanogen bromide-activated Sepharose resin using the protocol of the resin manufacturer (Amersham Pharmacia Biotech). The eluate was stored at 4 °C. SDS-polyacrylamide gel electrophoresis was performed using the protocol of Sambrook et al. (9$$Google Scholar). Western blot analysis was accomplished following the method of Ausubel et al. (10$$Google Scholar) with the following modifications; polyvinylidene difluoride membrane (Bio-Rad) was used for blotting, and the blot was probed with a 1:400 dilution of partially purified Piv antibody. The protein markers were SDS-PAGE standards, low range, or Kaleidoscope prestained standards (for Western blots) from Bio-Rad. DH5α containing pAG862, pAG862 and pAG607 or containing pAG862 and pMal-C2 were grown to OD600 = 0.6, induced with 0.05 mm IPTG, and incubated for 2 h at 37 °C. Plasmids were extracted using an alkaline lysis protocol (9$$Google Scholar) and digested with HindIII and KpnI. The restriction digests were examined on a 0.8% SeaKem (FMC Bioproducts) agarose gel. The DNA was visualized by ethidium bromide staining and documented by digital imaging of the UV-illuminated gel. Double-stranded DNA oligonucleotides, INV and SUB (see “Results”), were labeled with [γ-32P]ATP using T4 polynucleotide kinase following the protocols of Sambrook et al. (9$$Google Scholar). pAG604 and pAG605 were digested with SpeI andEcoRV to obtain 298-bp fragments containing thesub1 site to be used in DNase I protection assays. These fragments were labeled at the SpeI site with [α-32P]ATP using Klenow (exo−) DNA polymerase under conditions recommended by the manufacturer with the modification of incubating the reactions at 25 °C; the labeled fragments were electrophoresed on a 5% polyacrylamide gel, electroeluted from the gel, and stored at −20 °C. The pMxL1-dL24 plasmid, a deletion derivative of pMxL1, provided by C. Marrs (6Marrs C.F. Rozsa F.W. Hackel M. Stevens S.P. Glasgow A.C. J. Bacteriol. 1990; 172: 4370-4377Crossref PubMed Google Scholar), and the pMxL1invR fragment (obtained by PCR from pMxL1, usingPfu DNA polymerase and the primers: 5′-GCCAGCACGTGTCTAAAACTTACATTG and 5′-CCATACACCATCAGCAGCACG) were digested with various restriction enzymes as indicated under “Results.” The DNA fragments created were labeled with [α-32P]dATP or dCTP using Klenow (exo−) polymerase, gel-purified, and stored at −20 °C. Gel electrophoresis retardation, or gel shift, assays were initiated by mixing MBP-Piv protein with each radiolabeled DNA fragment (5 × 10−12m) in 1× binding buffer: 80 mm KCl, 20 mm Tris-Cl, pH 7.6, 5 mmCaCl2, 250 mg/ml poly(C), 1 mm dithiothreitol, and 50 mg/ml bovine serum albumin (NEB). The reactions were incubated for 20 min at room temperature, immediately loaded onto a nondenaturing polyacrylamide gel, and run at 4 °C in 0.5× TBE buffer. For the competition assays, increasing amounts of competitor DNA ranging from 1 to 500 molar excess was added to the reaction before addition of the protein. The specific DNA competitors were the SUB and INV double-stranded oligonucleotides and the nonspecific DNA competitor was a 39-bp oligonucleotide: 5′-TTAAGATCGATGACGTCAGATCTGAGCTCGATACTCGAG annealed to 5′-CTCGAGTATCGAGCTCAGATCTGACGTCATCGATC. For the DNase I protection assays performed at the sub1 site, the 298-bp fragments from pAG604 and pAG605, labeled on the top strand and bottom strand for sub1 (5 × 10−10m), were incubated for 20 min at room temperature with or without MBP-Piv (7 × 10−6m) in 1× binding buffer. DNase I protection assays were performed in binding buffer plus 5 mm MgCl2 with 0.02 units of DNase I as described previously (11Glasgow A.C. Bruist M.F. Simon M.I. J. Biol. Chem. 1989; 264: 10072-10082Abstract Full Text PDF PubMed Google Scholar). Chemical cleavage using the Maxam and Gilbert sequencing reactions A/G and C/T were performed with the same labeled fragments (12Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9012) Google Scholar). The footprinting and sequencing reactions were run on a 6% sequencing polyacrylamide gel. At the site of inversion, dimethyl sulfate interference assays were performed as described previously (16Koo H.S. Drak J. Rice J.A. Crothers D.M. Biochemistry. 1990; 29: 4227-4234Crossref PubMed Scopus (209) Google Scholar). Indirect DNase I and 1,10-phenanthroline-copper protection assays were also performed at the site of inversion following the protocol described by Kuwabara and Sigman (13Kuwabara M.D. Sigman D.S. Biochemistry. 1987; 26: 7234-7238Crossref PubMed Scopus (200) Google Scholar) with the following modifications for the DNase I protection assay: gel slices were treated with DNase I in 1× binding buffer plus 5 mm MgCl2 for 5–8 min before quenching the reaction with the addition of 20 mm EDTA. Gel electrophoresis retardation assays were performed with labeled DNA fragments (5 × 10−12m) and increasing amounts of MBP-Piv protein ranging from 0.6 to 1582 nm for binding to SUB and 0.6 to 4560 nm for binding to INV. Binding of MBP-Piv (P) to each DNA binding site (X) is represented by the equation:K = [P]n[X]/[Pn X], which translates into: ln([Pn X]/[X]) = nln[P] − ln[K]. [P] is the concentration of MBP-Piv monomers, [Pn] is the concentration of active MBP-Piv oligomers, n is the number of active monomers in the oligomer, [Pn X] is the concentration of bound DNA, and [X] is the concentration of unbound DNA. A plot of ln[P] versusln([Pn X]/[X]) gives a line with slope n (14Kristensen H.H. Valentin-Hansen P. Sogaard-Andersen L. J. Mol. Biol. 1996; 260: 113-119Crossref PubMed Scopus (19) Google Scholar). The amount of bound and unbound DNA was quantitated using a PhosphorImager: 445SI with ImageQuant software (Molecular Dynamics). Binding assays were performed in 1× binding buffer. Labeled, double-stranded oligonucleotide (5 × 10−12m) was incubated with MBP-Piv protein (6 × 10−7m) as indicated for 20 min at room temperature. Unlabeled, competitor oligonucleotide (2.5 × 10−8m) was added at time zero. At the indicated times, 20-μl aliquots were loaded onto a 10% nondenaturing polyacrylamide gel running at 120 V. Following electrophoresis, the gels were dried and imaged with a PhosphorImager: 445SI. ImageQuant software was used to quantitate the label in the bands corresponding to MBP-Piv-DNA complexes and unbound DNA. The fraction of the total DNA in the reaction that was bound by MBP-Piv was calculated by dividing the amount of labeled DNA present in the shifted complexes by the total labeled DNA in the reaction (i.e. the combined complexed and unbound DNA fragments quantitated for each reaction). The percent bound DNA was plotted as a function of the time after addition of competitor DNA. The half-life for each complex was the time required for a 50% reduction in protein-DNA complex due to dissociation of the MBP-Piv DNA complex. Based on the sequence of the piv gene, the Piv protein is predicted to be a 322-amino acid polypeptide with aM r of 36,935 and an isoelectric point of 10.72. To facilitate purification of Piv, M. lacunata piv was introduced into the expression vector pMal-C2, fusing malEto the 5′ end of piv (pAG607) to produce the fusion protein, MBP-Piv. MBP-Piv (79 kDa) was soluble and expressed at high levels. Affinity chromatography (amylose resin) was used to purify the MBP-Piv fusion protein for use in DNA binding assays. Fig.1 A shows a Coomassie-stained SDS-PAGE gel containing samples from a crude lysate used for purification of MBP-Piv and fractions collected from the 10 mm maltose elution of protein bound to the amylose column. ImageQuant analysis of a digital image of a Coomassie-stained, SDS-polyacrylamide gel containing serial dilutions of the purified protein indicated that the fractions used in all the following experiments are approximately 85% full-length MBP-Piv protein (data not shown). Fig. 1 B shows Western blot analysis of expression of the MBP-Piv fusion protein from pAG607 using polyclonal antisera raised against MBP-Piv and affinity-purified to obtain anti-Piv antisera as described under “Experimental Procedures.” MBP-Piv could be detected in the soluble fraction of a crude lysate prepared from DH5α cells containing pAG607 after induction with 0.5 mm IPTG (Fig. 1 B, lanes 3 and 4). The affinity-purified antibodies are specific for Piv as no cross-reactivity with MBP alone was observed (Fig. 1 B,lane 2). Cleavage of MBP-Piv with the protease factor Xa, which recognizes a specific cleavage site between MBP and Piv in the fusion protein, resulted in cleavage at the fusion junction sequence and, unfortunately, also within Piv itself. Because this additional cleavage site prevented separation of the full-length Piv protein from MBP by Factor Xa treatment, we determined whether the MBP-Piv fusion protein could carry out the functions of wild type Piv. An in vivo inversion assay was used to qualitatively estimate recombinase activity as an indication of functional protein-DNA interactions (Fig. 2). DH5α was cotransformed with a DNA inversion test plasmid, pAG862, and expression plasmids encoding either MBP alone (pMal-C2), or MBP-Piv (pAG607). After overnight expression of MBP or MBP-Piv, recombination of the invertible segment on pAG862 was assayed by restriction enzyme digestion of isolated plasmid DNA. In the original orientation of the test plasmid, digestion with HindIII and KpnI results in two bands, of 5.7 and 1.0 kb. If there is MBP-Piv-mediated inversion, the same digestion will yield two different unique bands, of 4.1 and 2.6 kb. With MBP alone, no inversion products are seen, whereas, in the presence of MBP-Piv, approximately 50% of the test plasmid is in the inverted orientation (Fig. 2). In a similar inversion assay, wild type Piv also directed inversion to yield approximately 50% of either orientation, representing an equilibrium point in the inversion reaction (data not shown). Thus, these results indicated that the MBP-Piv fusion is a functional recombinase; consequently, the uncleaved MBP-Piv fusion protein was used in subsequent in vitro DNA binding studies. Based on the binding specificity of other site-specific recombinases and transposases (reviewed in Refs. 1Craig N.L. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, DC1996: 2339-2362Google Scholar and 2Hallet B. Sherratt D.J. FEMS Microbiol Rev. 1997; 21: 157-178Crossref PubMed Google Scholar), we expected that Piv would bind DNA sequences encoding the cross-over sites for inversion (invL and invR) as well as possible accessory sites involved in recombination or regulation ofpiv expression. An assay for Piv DNA binding sites within the inversion region was carried out using gel electrophoresis mobility shift assays (EMSA; data not shown) with a series of restriction fragments that spanned both the invertible segment and the adjacent sequences required for inversion in E. coli (Fig.3 A). Several of the fragments were shifted in the presence of MBP-Piv (* in Fig. 3 A). Two fragments, 430 and 870 bp, were chosen for further analysis by DNase I footprinting. The 430-bp fragment was selected because it spans the left recombination site of the invertible element (invL). The 870-bp fragment also contains the invL recombination site, plus a potential accessory site (see below). DNase I protection assays with MBP-Piv and the 870-bp fragment showed protections and enhancements from about −285 to −265 bp upstream of the invL cross-over site (data not shown). However, due to the relatively large size of the fragment, the DNase I cleavage pattern at the site was not easily resolved. Therefore, a smaller fragment of 298 bp, containing the protected DNA sequence, was examined by DNase I protection assays (Fig. 3, B and C). The DNase I cleavage pattern in the MBP-Piv binding region has long tracts of no cleavage due to A-tract repeats that narrow the minor groove, thus inhibiting DNase I binding and cleavage (15Drew H.R. Travers A.A. Cell. 1984; 37: 491-502Abstract Full Text PDF PubMed Scopus (411) Google Scholar). These A-tracts are also appropriately phased along the DNA helix to contribute sequence-directed DNA bending to this region of the DNA (16.). Protections and enhancements from −233 to −247 and from −266 to −285 (Fig. 3, B and C) could be resolved in these DNase I protection assays. No other protected regions were detected on the 298-bp fragment. This MBP-Piv binding site is probably too distant from the site of DNA cleavage (+1) and strand exchange in the inversion reaction to be the binding site for the Piv subunit that actually catalyzes the recombination reaction. However, it is likely that this Piv binding site is an accessory site utilized in the assembly of an active synaptic complex for Piv-mediated DNA inversion (see “Discussion”). The 430-bp fragment (Fig. 3 A) contains the 26-bp site of inversion (invL) that was defined based upon sequence homology at the ends of the invertible segment (17Fulks K.A. Marrs C.F. Stevens S.P. Green M.R. J. Bacteriol. 1990; 172: 310-316Crossref PubMed Google Scholar). The initial EMSA analysis of MBP-Piv binding to the 430-bp fragment indicated that MBP-Piv does bind specifically, albeit weakly, to a sequence within this fragment (data not shown). However, DNase I and 1,10-phenanthroline-copper protection assays with the 430-bp fragment and MBP-Piv showed no evidence of protein-DNA interactions (data not shown). Because MBP-Piv does shift the 430-bp fragment in EMSA gels, indirect DNase I protection assays were performed with the 430-bp fragment in which the electrophoretically separated protein-DNA complex and unbound DNA are treated with DNase I in the non-denaturing polyacrylamide gel and then isolated from gel slices for electrophoresis on the DNA sequencing polyacrylamide gel (see “Materials and Methods”). Even though this assay should have enriched for DNA complexed with MBP-Piv, no protection of DNA sequence by MBP-Piv was observed. These results suggested that the binding of MBP-Piv to the 430-bp fragment was either unstable or not specific. To determine if the poor interactions of MBP-Piv with invLare due to the MBP protein at the amino terminus of Piv, Piv was tagged at the COOH terminus with six histidines (see “Materials and Methods”). The Piv-His6 fusion protein was partially purified and used in EMSA and DNase I protection assays with the 430-bp fragment and also with the 298-bp fragment containing the accessory Piv binding site (data not shown). All of the DNA binding results with Piv-His6 corresponded exactly with those obtained with MBP-Piv, indicating that the poor binding of MBP-Piv to invLis not due to steric hindrance by MBP of Piv-specific binding. Therefore, stable binding of Piv may require other factors such as accessory DNA binding sites, accessory proteins, and/or supercoiled DNA substrate to facilitate binding to the recombination sites (see “Discussion”). To characterize the interactions of MBP-Piv with the isolated invL and upstream accessory site, double-stranded oligonucleotides corresponding to each site were synthesized (designated INV and SUB, respectively; Fig. 4 A). The sequence of INV included the 26-bp region of homology from either end of the invertible element and 14 bp of sequence immediately upstream and downstream from the invL site. The DNA sequence of SUB (orstrong upstream binding site oligonucleotide) was designed based on the DNase I footprint originally obtained with the 870-bp DNA fragment (i.e. the protected region −269 to −284, but not the −233 to −245 region). The INV and SUB DNA binding substrates were used in an EMSA to examine MBP-Piv binding at each site (Fig. 4 B). Two shifted complexes (S-1 and S-2,lane 2, Fig. 4 B) were formed with the SUB oligonucleotide, indicating that at least two MBP-Piv proteins bind SUB. Because the SUB oligonucleotide does not include the DNA sequence from −233 to −247 bp (see Fig. 3), we have designated the MBP-Piv binding site encoded within the SUB oligonucleotide,sub1. MBP-Piv binding to INV resulted in one primary complex (I-1) that migrated at approximately the same rate as the slower migrating complex formed with SUB (S-2). Because the difference in the molecular weights of the INV and SUB oligonucleotides is nominal in comparison to the MBP-Piv molecular weight, this result suggests that the I-1 and S-2 protein-DNA complexes contain the same number of MBP-Piv protomers. No faster migrating protein-DNA complex was seen with INV when the concentration of MBP-Piv was decreased (data not shown). The presence of a slower complex (I-2) was detected with some variability in the EMSAs. The formation of this complex suggests that a higher multimeric MBP-Piv may bind INV or an additional INV oligonucleotide may bind the I-1 complex. Based upon the predicted molecular weights of INV, SUB, and MBP-Piv, the electrophoretic mobilities of I-1, S-1, and S-2 indicate that INV is bound by at least two MBP-Piv protomers while SUB is bound by either single (S-1) or multiple (S-2) MBP-Piv protomers. Comparison of the Piv DNA binding substrates, INV and SUB, revealed very little homology (TATNC), suggesting that Piv has two different DNA recognition sites. To further examine the interactions of MBP-Piv with the apparently different binding sites on INV and SUB, competition assays were performed using each of the oligonucleotides as both binding substrate and competitor DNA. In Fig. 5 A, the SUB oligonucleotide was labeled and used in DNA binding assays with MB
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