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- W2085491689 abstract "Assembly of P1 plasmid partition complexes at the partition site, parS, is nucleated by a dimer of P1 ParB and Escherichia coli integration host factor (IHF), which promotes loading of more ParB dimers and the pairing of plasmids during the cell cycle. ParB binds several copies of two distinct recognition motifs, known as A- and B-boxes, which flank a bend in parS created by IHF binding. The recent crystal structure of ParB bound to a partial parS site revealed two relatively independent DNA-binding domains and raised the question of how a dimer of ParB recognizes its complicated arrangement of recognition motifs when it loads onto the full parS site in the presence of IHF. In this study, we addressed this question by examining ParB binding activities to parS mutants containing different combinations of the A- and B-box motifs in parS. Binding was measured to linear and supercoiled DNA in electrophoretic and filter binding assays, respectively. ParB showed preferences for certain motifs that are dependent on position and on plasmid topology. In the simplest arrangement, one motif on either side of the bend was sufficient to form a complex, although affinity differed depending on the motifs. Therefore, a ParB dimer can load onto parS in different ways, so that the initial ParB-IHF-parS complex consists of a mixture of different orientations of ParB. This arrangement supports a model in which parS motifs are available for interas well as intramolecular parS recognition. Assembly of P1 plasmid partition complexes at the partition site, parS, is nucleated by a dimer of P1 ParB and Escherichia coli integration host factor (IHF), which promotes loading of more ParB dimers and the pairing of plasmids during the cell cycle. ParB binds several copies of two distinct recognition motifs, known as A- and B-boxes, which flank a bend in parS created by IHF binding. The recent crystal structure of ParB bound to a partial parS site revealed two relatively independent DNA-binding domains and raised the question of how a dimer of ParB recognizes its complicated arrangement of recognition motifs when it loads onto the full parS site in the presence of IHF. In this study, we addressed this question by examining ParB binding activities to parS mutants containing different combinations of the A- and B-box motifs in parS. Binding was measured to linear and supercoiled DNA in electrophoretic and filter binding assays, respectively. ParB showed preferences for certain motifs that are dependent on position and on plasmid topology. In the simplest arrangement, one motif on either side of the bend was sufficient to form a complex, although affinity differed depending on the motifs. Therefore, a ParB dimer can load onto parS in different ways, so that the initial ParB-IHF-parS complex consists of a mixture of different orientations of ParB. This arrangement supports a model in which parS motifs are available for interas well as intramolecular parS recognition. In bacterial cells, the dynamic localization of low copy number plasmids is directed by their partition systems, which ensure the proper segregation and thus stable inheritance of these plasmids in growing cell populations. The P1 plasmid exists as a stable, autonomously replicating, low copy number plasmid in Escherichia coli, and its partition system has served as a paradigm for homologous systems found in many naturally occurring bacterial plasmids as well as several bacterial chromosomes (reviewed in Refs. 1Hayes F. Barilla D. Nat. Rev. Microbiol. 2006; 4: 133-143Crossref PubMed Scopus (114) Google Scholar and 2Funnell B.E. Slavcev R.A. Funnell B.E. Phillips G.J. Plasmid Biology. ASM Press, Washington, D. C.2004: 81-103Google Scholar). These partition systems typically consist of two proteins, ParA and ParB, that act on a plasmid partition site, parS (P1 nomenclature). The partition reaction occurs through a series of DNA-protein and protein-protein interactions that lead to P1 plasmid localization within an E. coli cell. ParB acts as the key partition site binding protein (3Davis M.A. Austin S. EMBO J. 1988; 7: 1881-1888Crossref PubMed Scopus (97) Google Scholar, 4Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6657-6661Crossref PubMed Scopus (77) Google Scholar). ParB initially loads onto parS as a dimer and then recruits multiple dimers to form a large nucleoprotein complex, which is proposed to mediate P1 plasmid pairing through ParB-ParB interactions (5Bouet J.-Y. Surtees J.A. Funnell B.E. J. Biol. Chem. 2000; 275: 8213-8219Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Erdmann N. Petroff T. Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14905-14910Crossref PubMed Scopus (92) Google Scholar, 7Edgar R. Chattoraj D. Yarmolinsky M. Mol. Microbiol. 2001; 42: 1363-1370Crossref PubMed Scopus (63) Google Scholar). ParA is an ATPase that interacts with this complex and through an unknown process, mediates the specific localization of P1 plasmids (6Erdmann N. Petroff T. Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14905-14910Crossref PubMed Scopus (92) Google Scholar, 8Davis M.A. Radnedge L. Martin K.A. Hayes F. Youngren B. Austin S.J. Mol. Microbiol. 1996; 21: 1029-1036Crossref PubMed Scopus (74) Google Scholar, 9Bouet J.-Y. Funnell B.E. EMBO J. 1999; 18: 1415-1424Crossref PubMed Scopus (138) Google Scholar, 10Li Y.F. Austin S. Mol. Microbiol. 2002; 46: 63-74Crossref PubMed Scopus (61) Google Scholar). The only host factor known to participate in P1 partition is E. coli integration host factor (IHF), 2The abbreviations used are: IHF, integration host factor; HTH, helix-turn-helix. which assists ParB in the initial DNA binding step by greatly increasing the affinity of ParB for parS (4Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6657-6661Crossref PubMed Scopus (77) Google Scholar). ParB is an unusual site-specific DNA-binding protein in that it recognizes two distinct sequence motifs, called A-boxes (ATTTCAA/C) and B-boxes (TCGCCA), in parS (11Davis M.A. Martin K.A. Austin S.J. EMBO J. 1990; 9Crossref Scopus (26) Google Scholar, 12Funnell B.E. Gagnier L. J. Biol. Chem. 1993; 268: 3616-3624Abstract Full Text PDF PubMed Google Scholar). There are multiple copies of these boxes in parS, and they are asymmetrically arranged around an IHF binding site (Fig. 1). The spacing and orientation of these binding motifs are critical for complex formation in vitro as well as parS activity in vivo (12Funnell B.E. Gagnier L. J. Biol. Chem. 1993; 268: 3616-3624Abstract Full Text PDF PubMed Google Scholar, 13Hayes F. Austin S.J. J. Mol. Biol. 1994; 243: 190-198Crossref PubMed Scopus (36) Google Scholar). IHF bends parS, which allows ParB to contact its specific binding motifs flanking the bend, resulting in a high-affinity protein-DNA complex. In the absence of IHF, ParB binds specifically but more weakly to parS and requires only the right half of parS for activity. In vivo, a 22-bp sequence on the right side of parS (boxes A2-A3-B2, called parS-small in Fig. 1) is sufficient but not optimal for partition (14Martin K.A. Davis M.A. Austin S. J. Bacteriol. 1991; 173: 3630-3634Crossref PubMed Google Scholar). The optimal parS site extends from box B1 to box B2 and requires IHF. All motifs except boxes A1 and A4 are necessary for wild-type parS activity in vivo (11Davis M.A. Martin K.A. Austin S.J. EMBO J. 1990; 9Crossref Scopus (26) Google Scholar, 12Funnell B.E. Gagnier L. J. Biol. Chem. 1993; 268: 3616-3624Abstract Full Text PDF PubMed Google Scholar, 13Hayes F. Austin S.J. J. Mol. Biol. 1994; 243: 190-198Crossref PubMed Scopus (36) Google Scholar). Interestingly, however, the position and orientation of boxes A1 and A4 have been conserved in a variety of related parS sites (2Funnell B.E. Slavcev R.A. Funnell B.E. Phillips G.J. Plasmid Biology. ASM Press, Washington, D. C.2004: 81-103Google Scholar). Biochemical assays have shown that the C-terminal half of ParB (residues 142–333) contains all of the information necessary to form the dimeric, high-affinity (IHF-stimulated) ParB complex at parS (15Surtees J.A. Funnell B.E. J. Biol. Chem. 2001; 276: 12385-12394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Recently, the crystal structure of ParB-(142–333) bound to a 25-bp DNA duplex containing the parS-small sequence has been determined (16Schumacher M.A. Funnell B.E. Nature. 2005; 438: 516-519Crossref PubMed Scopus (94) Google Scholar). ParB contains two essentially independent DNA-binding domains (a helix-turn-helix (HTH) domain between residues 147–270 and a dimer domain between residues 275 and 333) separated by a short (4-residue) flexible linker. This arrangement permits ParB to contact a variety of A- and B-box combinations within parS. As functional studies predicted, the crystal structure of ParB-(142–333) confirms that the HTH motif binds the A-boxes within parS and the dimerization domain binds the B-boxes (15Surtees J.A. Funnell B.E. J. Biol. Chem. 2001; 276: 12385-12394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16Schumacher M.A. Funnell B.E. Nature. 2005; 438: 516-519Crossref PubMed Scopus (94) Google Scholar, 17Radnedge L. Davis M.A. Austin S.J. EMBO J. 1996; 15: 1155-1162Crossref PubMed Scopus (42) Google Scholar, 18Hayes F. Radnedge L. Davis M.A. Austin S.J. Mol. Microbiol. 1993; 11: 249-260Crossref Scopus (55) Google Scholar). The dimerization interaction creates a novel DNA binding motif that requires contributions from each monomer to bind a box B sequence. The arrangement of the HTH motifs is unusual in that they are on opposite sides of the dimer pointing away from each other so they cannot simultaneously contact a single inverted repeat. In fact, one ParB dimer contacts its different motifs on separate copies of the same 25-mer parS-small oligonucleotide, effectively bridging different DNA molecules. The bridging activity illustrates how ParB might contact separate motifs intramolecularly across an IHF-directed bend but also suggests that it may participate in pairing plasmids intermolecularly across parS sites (16Schumacher M.A. Funnell B.E. Nature. 2005; 438: 516-519Crossref PubMed Scopus (94) Google Scholar). We were interested in how ParB contacts the parS site, particularly across the IHF-directed bend and potentially across adjacent DNA molecules. Chemical footprinting experiments have shown that all A- and B-boxes within parS are protected by a dimer of ParB (5Bouet J.-Y. Surtees J.A. Funnell B.E. J. Biol. Chem. 2000; 275: 8213-8219Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), but the crystal structure implies that one dimer cannot contact all motifs simultaneously (16Schumacher M.A. Funnell B.E. Nature. 2005; 438: 516-519Crossref PubMed Scopus (94) Google Scholar). In this study we examined how a dimer of ParB recognizes the parS site. Do the different A- and B-boxes work together in providing a high-affinity binding site for ParB, or is there a distinct set of ParB-IHF-parS complexes that uses specific A- and B-box combinations? Does ParB have a binding preference for specific A- or B-boxes within parS? To address these questions, variants of parS were created with A- and B-boxes systematically mutated individually or in combination with each other. We found that ParB prefers different subsets of motifs when it initially binds to parS. Our results support the model that the arrangement of recognition motifs in parS is necessary to build the proper architecture of partition complexes at and across partition sites. Bacterial Strains and Plasmids—E. coli strain DH5 (F– endA1 hsdR17 (rK– mK+) supE44 thi-1 gyrA96 recA1) was used to construct and maintain plasmids. The plasmid pBend5, a derivative of pBend2 (19Kim J. Zwieb C. Wu C. Adhya S. Gene. 1989; 85: 15-23Crossref PubMed Scopus (321) Google Scholar), was used as the vector to create plasmids containing the parS variants, which were the DNA substrates for biochemical experiments. All parS variants used in this study were constructed by cloning complementary synthetic oligonucleotides (80–88-mers, depending on the variant; see supplemental Table 1) into the HpaI site of pBend5. The plasmid parS sequences were confirmed by DNA sequencing at York University (Toronto) and/or Macrogen Inc. (Seoul, South Korea). Reagents—Synthetic oligodeoxynucleotides were purchased from Invitrogen. Sources for other reagents were as follows: [α-32P]dCTP and S-[3H]adenosyl-l-methionine were from PerkinElmer; bovine serum albumin and salmon sperm DNA, from Sigma; restriction enzymes, Klenow DNA polymerase, and HaeIII methylase, from New England Biolabs. DNA and Proteins—For gel mobility shift assays, the DNA substrates were total XhoI restriction digests of plasmid DNA. DNA fragments were labeled at their 3′-ends with [α-32P]dCTP and DNA polymerase I (Klenow fragment), and then purified by phenol-chloroform extraction and ethanol precipitation steps. For nitrocellulose filter binding assays, supercoiled plasmid DNAs were purified in cesium chloride gradients, and labeled with S-[3H]adenosyl-l-methionine and HaeIII methylase as described (4Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6657-6661Crossref PubMed Scopus (77) Google Scholar). ParB (fraction V) was purified as described previously (9Bouet J.-Y. Funnell B.E. EMBO J. 1999; 18: 1415-1424Crossref PubMed Scopus (138) Google Scholar). IHF was purified essentially as described (20Nash H.A. Robertson C.A. Flamm E. Weisberg R.A. Miller H.I. J. Bacteriol. 1987; 169: 4124-4127Crossref PubMed Scopus (164) Google Scholar), except that the phosphocellulose step was followed by Mono-S chromatography in 15 mm phosphate buffer, pH 6.4, 5% glycerol, and protein was eluted with a 50 mm–1 m NaCl gradient in the same buffer. Gel Mobility Shift Assays—The standard reaction mixture (10 μl) contained 1 nm 32P-labeled DNA in 50 mm Hepes-KOH (pH 7.5), 150 mm KCl, 10% glycerol, 80 μg of bovine serum albumin/ml, and 250 μg of sonicated salmon sperm DNA/ml. IHF, when present, was included at 500 nm (so its binding would not be limiting). The mixtures were assembled on ice, incubated for 20 min at 30 °C, and analyzed by electrophoresis in 5% polyacrylamide gels in 90 mm Tris borate, 1 mm EDTA. Electrophoresis was performed at 150 V for 3.5 h at 4 °C. The gels were dried onto Whatman DE81 paper and exposed to a phosphor screen for imaging by a PhosphorImager. Data were quantified using ImageQuant software (GE Healthcare). For each lane in the gels, the radioactivity (as phosphorimaging counts) in the area corresponding to a dimeric ParB-IHF-parS complex was measured and expressed as a fraction of the value corresponding to the parS DNA fragment in the absence of ParB. Analysis of the Data—All variants were tested typically three to six times for binding by ParB over a range of ParB concentrations, and the results from these titrations were averaged. The error in individual values was typically less than 10% of substrate, ranging from 2 to 15% of substrate. When a dimer of ParB binds parS, the interaction occurs in an equimolar fashion. Therefore, the averaged data set from a particular variant was then fit to the Langmuir binding equation (21Reichheld S.E. Davidson A.R. J. Mol. Biol. 2006; 361: 382-389Crossref PubMed Scopus (20) Google Scholar, 22Larsson A. Axelsson B. J. Immunol. Methods. 1991; 137: 253-259Crossref PubMed Scopus (7) Google Scholar, 23Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar) using SigmaPlot 2000 software. I=I0+(I∞[B]/Kd)/(1+[B]/Kd)(Eq.1) The band intensity, I, is a function of the free ParB concentration [B] and the dissociation constant (Kd), and [B] is defined by the following equation, [B]=(-([S]-[BT]+Kd)+SQRT(([S]-[BT]+Kd)2+4*Kd*[BT]))/2(Eq.2) where [S] is the total concentration of parS substrate, [BT] is the total concentration of ParB, and SQRT is the square root of the values in parentheses. Not all A- and B-boxes Are Required for a Dimer of ParB to Recognize and Bind to parS—The first step in partition complex assembly is the binding of one ParB dimer to parS across the IHF-directed bend (5Bouet J.-Y. Surtees J.A. Funnell B.E. J. Biol. Chem. 2000; 275: 8213-8219Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We were interested in the nature of this protein-DNA interaction as well as the role of different motifs in partition complex function. Our recent crystal structure of ParB-(142–333) complexed with parS-small had raised questions about how ParB recognizes a full parS site (16Schumacher M.A. Funnell B.E. Nature. 2005; 438: 516-519Crossref PubMed Scopus (94) Google Scholar). We typically study the interaction by gel mobility shift assays in order to examine specific complexes and monitor the stoichiometry of assembly (5Bouet J.-Y. Surtees J.A. Funnell B.E. J. Biol. Chem. 2000; 275: 8213-8219Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In situ OP-Cu footprinting of the dimeric ParB-IHF-parS complex showed protection of all A- and B-boxes within parS, suggesting two possible mechanisms of ParB binding to parS: 1) a single dimer of ParB binds all or most of its recognition sequences on both the left and right arms of parS simultaneously; or 2) a dimer of ParB can bind various combinations of motifs across an IHF-directed bend, so that the initial complex actually represents a mixture of complexes with different modes of ParB binding to parS. To examine these possibilities, we created a spectrum of mutant sites in which A- or B-boxes were removed by substitution with a restriction site (Fig. 2). In our nomenclature, mutations are designated by the box name in lowercase (for example, parS[a2] lacks the box A2-specific sequence; Fig. 2). Substitution allowed for the removal of specific base pair contacts with ParB while maintaining the sequence, spacing, and orientation of the remaining boxes. The use of different restriction sites also marked each parS variant differently. We constructed a negative control, called parS[Δab], which lacks all A- and B-box sequences but retains the IHF binding site (Fig. 2). We defined parS+ as all sequences between and including box B1 to B2 because box A4 is dispensable for partition in vivo (13Hayes F. Austin S.J. J. Mol. Biol. 1994; 243: 190-198Crossref PubMed Scopus (36) Google Scholar, 24Funnell B.E. Gagnier L. Biochimie (Paris). 1994; 76: 924-932Crossref PubMed Scopus (22) Google Scholar), and its omission allowed us to reduce the number and complexity of variants that we created. We did subsequently confirm that box A4 was dispensable in our assays; we observed no quantitative differences in ParB binding affinity between parS+ and the parS+-A4 site (see below and Table 1).TABLE 1Summary of ParB binding activity to parS site variants in the presence of IHFparS variantBoxes remainingaDashes indicate the position of boxes that have been mutated (with respect to parS+).Kd(app)bKd(app) was calculated as described under “Experimental Procedures.” NB, no measurable binding activity. >1 μm, substrate binding below 50% at 1 μm ParB.nmparS+B1 A1 A2 A3 B217 ± 4Δab— — — — —NBb1— A1 A2 A3 B27 ± 2a1B1 — A2 A3 B2580 ± 40a2B1 A1 — A3 B210 ± 2a3B1 A1 A2 — B261 ± 25b2B1 A1 A2 A3 —58 ± 23b1,a1— — A2 A3 B2690 ± 210b1,a2— A1 — A3 B249 ± 20b1,a3— A1 A2 — B2119 ± 59b1,b2— A1 A2 A3 —56 ± 28a1,a2B1 — — A3 B2>1 μma1,a3B1 — A2 — B2NBa1,b2B1 — A2 A3 —NBa3,b2B1 A1 A2 — —59 ± 35a2,b2B1 A1 — A3 —400 ± 160a2,a3B1 A1 — — B2450 ± 150b1,a3,b2— A1 A2 — —28 ± 6b1,a2,b2— A1 — A3 —550 ± 180b1,a2,a3— A1 — — B2500 ± 220RevA1A2— 1A 2A — —NBparS+-A4B1 A1 A2 A3 B2 A414 ± 7b1,a2,a3,b2-A4— A1 — — — A4760 ± 210a1,a2,a3,b2-A4B1 — — — — A4NBa Dashes indicate the position of boxes that have been mutated (with respect to parS+).b Kd(app) was calculated as described under “Experimental Procedures.” NB, no measurable binding activity. >1 μm, substrate binding below 50% at 1 μm ParB. Open table in a new tab We first tested the effect of mutating individual motifs on the formation of the partition complex. The parS substrates were incubated with ParB and IHF, and the resulting complexes were separated by gel electrophoresis (Fig. 3). An apparent dissociation constant (Kd(app)) was then determined through quantitative analysis of ParB-IHF-parS complex formation (Table 1). The data were evaluated using the Langmuir formula (see “Experimental Procedures”), a common way of describing specific, high-affinity binding of two molecules (21Reichheld S.E. Davidson A.R. J. Mol. Biol. 2006; 361: 382-389Crossref PubMed Scopus (20) Google Scholar, 22Larsson A. Axelsson B. J. Immunol. Methods. 1991; 137: 253-259Crossref PubMed Scopus (7) Google Scholar, 23Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar). The half-interval method was then used on the fitted curves to obtain the Kd(app) value, where Kd(app) is defined as the amount of ParB necessary for 50% maximal binding. The theoretical Langmuir curves and experimental binding curves produced Kd(app) values that were very similar. The results showed that not all motifs are necessary for the initial recognition of parS by ParB, and that some motifs are more important than others (Figs. 3 and 4; Table 1). Mutation of box B1 or box A2 had no significant effect on complex formation compared with that with parS+. However, removal of the other three motifs did reduce ParB affinity for parS. Mutation of box A3 or box B2 each reduced complex formation by ∼3–4-fold. The removal of box A1 produced the greatest reduction in complex formation. The Kd(app) was 30-fold greater than when using parS+. Therefore in this assay box A1 is a stronger recognition sequence than box B1 for ParB to bridge over the IHF directed bend. Comparison of parS[a3]to parS[a2] indicates that box A3 is the preferred A-box on the right arm of parS. Finally, the data indicate that this initial ParB-IHF-parS complex is only a precursor of the functional partition complex, because not all motifs necessary for parS function are necessary to form this first complex. We also noted that ParB bound to all parS variants with the same stoichiometry as to parS+, as the mobility of the ParB-IHF-parS complexes were identical in all gels that we examined (Fig. 3). Motif Use Is Influenced by Substrate Topology—The effect of mutation of box A1 on complex formation was unexpected because genetic assays had shown that it is dispensable for parS activity in vivo (11Davis M.A. Martin K.A. Austin S.J. EMBO J. 1990; 9Crossref Scopus (26) Google Scholar). We confirmed that the single motif mutations we used in this study behaved identically in vivo to mutations published previously (12Funnell B.E. Gagnier L. J. Biol. Chem. 1993; 268: 3616-3624Abstract Full Text PDF PubMed Google Scholar, 13Hayes F. Austin S.J. J. Mol. Biol. 1994; 243: 190-198Crossref PubMed Scopus (36) Google Scholar, 24Funnell B.E. Gagnier L. Biochimie (Paris). 1994; 76: 924-932Crossref PubMed Scopus (22) Google Scholar) by using incompatibility assays, which measure the ability of parS to destabilize a miniP1 partitioned by parS+. The parS[b1], parS[a2], parS[a3], and parS[b2] substitutions destroyed parS activity (were Inc–) compared with parS[a1], which behaved as parS+ (Inc+; data not shown). We considered the possibility that DNA topology was important because plasmid DNA would normally be negatively supercoiled in vivo. We used nitrocellulose filter binding assays with 3H-labeled plasmid DNA in vitro to test the influence of mutations in each motif on ParB binding to supercoiled substrates (Fig. 5). This assay is effective in examining ParB binding directly because under these conditions IHF binds DNA to nitrocellulose poorly (4Funnell B.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6657-6661Crossref PubMed Scopus (77) Google Scholar). In contrast to the assays on linear DNA, ParB bound parS+, parS[a1], and parS[b1] to similar extents in this assay (Fig. 5A). The mutations in the right side of parS, however, had effects on supercoiled DNA similar to those they manifested on linear DNA. Mutation of box A2 did not affect ParB binding activity, whereas mutation of box A3 or box B2 reduced ParB affinity for these parS variants. Because removing either one of the left-side boxes did not significantly affect binding, we removed both boxes B1 and A1 (Fig. 5A). The ParB binding activity to parS[b1,a1] was greatly reduced; therefore either box B1 or box A1 on the left arm of parS is necessary for this complex, but either one will suffice in this assay. This result confirms that the complexes measured in these filter-binding assays are using recognition motifs on both the left and right arms of parS over an IHF-directed bend, and thus the use of box B1 by ParB is strongly influenced by the topological context of the parS site. Therefore the ability to use box B1 is dependent on negative supercoiling, but in plasmids with this topology, either box A1 or B1 is sufficient to bridge across the IHF-directed bend. The role of Right-side Motifs in ParB Recognition—We next examined parS variants lacking two motifs (Fig. 2). First, each right-side motif was mutated in combination with either box A1 or box B1 (Fig. 6A and Table 1). All double mutants lacking A1 were poor substrates for ParB. parS sites lacking box B1 and one right-side motif were similar to or slightly weaker than the corresponding parS sites with single right-side mutations. Therefore two motifs on the right side are sufficient to mediate a relatively strong interaction with ParB as long as there is a specific left-side interaction. We concluded that box B1 could not contribute significantly because the substrates were linear. We confirmed this conclusion by testing two of these right-side mutations (a2 or a3) in the presence of either a1 or b1 substitutions in parS sites on supercoiled plasmids in nitrocellulose filter binding assays. In this experiment, parS[a1,a2] behaved as parS[b1,a2], and parS[a1,a3] behaved as parS[b1,a3] (Fig. 5B). Therefore, as seen with the single mutants, either box B1 or A1 is sufficient for a specific interaction of ParB with the left side of parS on supercoiled substrates. Because, with the exception of box A1, the gel-shift and filter-binding assays showed similar motif preferences for ParB, we continued the analysis using gel electrophoresis, as it allowed us to identify and quantify the dimeric ParB-IHF-parS complexes. We next examined the behavior of parS sites with double mutations in the right side of parS (Fig. 6B) to see whether any of the three right-side motifs were sufficient to mediate a specific interaction with ParB and, if so, whether they were equivalent. The answer was yes, all could interact specifically with ParB, but no, they were not equivalent. parS sites with only box A3 or box B2 were weaker, but specific sites (parS[a2,b2] and parS[a2,a3] were better substrates than parS[Δab]; Fig. 6B). However the parS site with only box A2 (parS[a3,b2]; Fig. 6B) was similar to parS sites lacking only one motif on the right side of parS (Fig. 6A; Table 1). We think that the simplest interpretation of these combinations is that in the presence of IHF and a specific left-side interaction, box A2 alone is sufficient to mediate a relatively strong interaction on the right side of parS. However in the absence of box A2, both boxes A3 and B2 must be present for highest binding activity of the right side of parS (Fig. 6B and Table 1). Finally, we tested parS variants that contained only motifs A1 and A2 (parS[b1,a3,b2]), only A1 and A3 (parS[b1,a2,b2]), and only A1 and B2 (parS[b1,a3,b2]) to confirm that only one motif on each side of parS was sufficient to mediate a specific interaction. Within the limits of experimental error, the affinity of ParB for these parS variants was similar to that when box B1 was present (Fig. 6B; Table 1). Our results indicating a role for box A1 on linear DNA prompted us to reassess the involvement of box A4 in complex formation, even though the presence of box A4 did not affect the activity of parS+ (Figs. 3 and 4, Table 1). We asked whether box A4 could substitute as a single motif on the right side of parS. ParB bound weakly to parS, with only boxes A1 and A4 (Fig. 6B; Table 1), and not to parS with boxes B1 and A4. Note that box A4 is 22 bp, or two turns of the helix, to the right of box A2, so we interpret the A1–A4 activity as ParB bridging across the bend in a specific and similar, but weaker, fashion than as between A1 and A2. In summary, the parS variants that we constructed for this analysis fell into three general categories: strong binding sites (with Kd(app) values similar to or only a fewfold higher than parS+); weak but specific binding sites (such as parS[a2,b2]); and sites with no detectable specific binding (equivalent to parS[Δab]). Several representative parS sites are graphed over a large titration range of ParB to illustrate these groups in Fig. 6C. All parS sites with intact A1 boxes fell into the former two categories (with linear DNA), indicating that bridging across the IHF-directed bend could occur with a minimum of one motif on each side of parS. ParB Interactions across the Bend in parS—All of the previous assays included IHF, and thus we have not directly measured the role of the IHF-directed bend. To ask how ParB binds to parS in the absence of the bend, we measured ParB interactions with parS sites lacking one or more motifs in the absence of IHF (Fig. 7A). Consistent with previous observations that the right side of parS, or parS-small, is the minimal partition site, removal of motifs on the left side of parS did not affect ParB binding in the absence of IHF. In other words, without the bend, ParB does not interact productively with the left side of parS. Examina" @default.
- W2085491689 created "2016-06-24" @default.
- W2085491689 creator A5051540459 @default.
- W2085491689 creator A5060040224 @default.
- W2085491689 creator A5060132420 @default.
- W2085491689 date "2007-04-01" @default.
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- W2085491689 title "P1 Partition Complex Assembly Involves Several Modes of Protein-DNA Recognition" @default.
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