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• W2046332670 abstract "During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 Å from its normal position to bind in a cytosine-specific pocket on the surface of Tus. During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 Å from its normal position to bind in a cytosine-specific pocket on the surface of Tus. In most bacterial species, chromosomal DNA replication initiates at a unique origin (oriC in Escherichia coli), and it proceeds bidirectionally until the two replication forks meet in the terminus region located opposite the origin (Figure 1A). The E. coli terminus contains ten 23 bp Ter sites (TerA–J) arranged in two oppositely oriented groups of five (Coskun-Ari and Hill, 1997Coskun-Ari F.F. Hill T.M. Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.J. Biol. Chem. 1997; 272: 26448-26456Crossref PubMed Scopus (47) Google Scholar). Each of the Ter sites binds Tus, the 36 kDa terminator protein (Hill et al., 1989Hill T.M. Tecklenburg M.L. Pelletier A.J. Kuempel P.L. tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNA-binding protein.Proc. Natl. Acad. Sci. USA. 1989; 86: 1593-1597Crossref PubMed Scopus (69) Google Scholar, Hidaka et al., 1989Hidaka M. Kobayashi T. Takenaka S. Takeya H. Horiuchi T. Purification of a DNA replication terminus (ter) site-binding protein in Escherichia coli and identification of the structural gene.J. Biol. Chem. 1989; 264: 21031-21037Abstract Full Text PDF PubMed Google Scholar, Hill, 1992Hill T.M. Arrest of bacterial DNA replication.Annu. Rev. Microbiol. 1992; 46: 603-633Crossref PubMed Scopus (78) Google Scholar, Kamada et al., 1996Kamada K. Horiuchi T. Ohsumi K. Shimamoto N. Morikawa K. Structure of a replication-terminator protein complexed with DNA.Nature. 1996; 383: 598-603Crossref PubMed Scopus (87) Google Scholar; reviewed in Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and anti-helicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (103) Google Scholar). Thus the first of the two forks to arrive in the terminus region encounters the same face of Tus bound at each of the first series of Ter sites, termed the permissive face. It apparently displaces the bound Tus and passes through to the second series, where it now encounters a nonpermissive face, and its progress is blocked: the fork is trapped between oppositely oriented Ter sites (Hill et al., 1987Hill T.M. Henson J.M. Kuempel P.L. The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication.Proc. Natl. Acad. Sci. USA. 1987; 84: 1754-1758Crossref PubMed Scopus (71) Google Scholar, Hill, 1992Hill T.M. Arrest of bacterial DNA replication.Annu. Rev. Microbiol. 1992; 46: 603-633Crossref PubMed Scopus (78) Google Scholar). Since the ten Ter sites have no inverted symmetry of sequence or direct repeats (Figure 1B), and Tus is a monomeric protein that forms a simple 1:1 complex with them (Coskun-Ari et al., 1994Coskun-Ari F.F. Skokotas A. Moe G.R. Hill T.M. Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli.J. Biol. Chem. 1994; 269: 4027-4034Abstract Full Text PDF PubMed Google Scholar), this evident polarity of fork arrest cannot be due to Tus acting as a simple thermodynamic clamp. Moreover, despite knowledge of the crystal structure of a Tus-TerA complex (Kamada et al., 1996Kamada K. Horiuchi T. Ohsumi K. Shimamoto N. Morikawa K. Structure of a replication-terminator protein complexed with DNA.Nature. 1996; 383: 598-603Crossref PubMed Scopus (87) Google Scholar) and extensive work on the process of replication fork arrest and the stability of complexes of Tus with variant Ter sites, there has been no satisfactory explanation of the mechanism that determines polarity of the Tus-Ter block (reviewed by Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and anti-helicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (103) Google Scholar). This mechanism is resolved in the present work. The X-ray crystal structure of the Tus-TerA complex showed that many of the conserved residues among the various Ter sites make base-specific contacts with the protein (Kamada et al., 1996Kamada K. Horiuchi T. Ohsumi K. Shimamoto N. Morikawa K. Structure of a replication-terminator protein complexed with DNA.Nature. 1996; 383: 598-603Crossref PubMed Scopus (87) Google Scholar, Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and anti-helicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (103) Google Scholar). The dissociation constant (KD) of the Tus-TerB complex was reported to be 0.3 pM in 0.15 M potassium glutamate, and the half-life of the complex was 550 min (Gottlieb et al., 1992Gottlieb P.A. Wu S. Zhang X. Tecklenburg M. Kuempel P. Hill T.M. Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.J. Biol. Chem. 1992; 267: 7434-7443Abstract Full Text PDF PubMed Google Scholar). This is therefore the most stable complex known of a monomeric, sequence-specific, DNA binding protein with its double-stranded recognition sequence. As expected for a complex in which many interactions are electrostatic (Kamada et al., 1996Kamada K. Horiuchi T. Ohsumi K. Shimamoto N. Morikawa K. Structure of a replication-terminator protein complexed with DNA.Nature. 1996; 383: 598-603Crossref PubMed Scopus (87) Google Scholar), binding is strongly dependent on ionic strength, with the value of KD rising to about 1 nM and the half-life decreasing to about 2 min in a buffer containing 0.25 M KCl, as assessed by surface plasmon resonance (SPR) studies (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar). Complexity beyond simple DNA binding in determining the polarity of fork arrest is suggested by identification of mutants of TerB that bind Tus as strongly as the wild-type but that are defective in replication fork arrest in vivo (Coskun-Ari and Hill, 1997Coskun-Ari F.F. Hill T.M. Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.J. Biol. Chem. 1997; 272: 26448-26456Crossref PubMed Scopus (47) Google Scholar) and mutants of Tus that are more defective in fork arrest than DNA binding (Skokotas et al., 1995Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.J. Biol. Chem. 1995; 270: 30941-30948Crossref PubMed Scopus (13) Google Scholar, Henderson et al., 2001Henderson T.A. Nilles A.F. Valjavec-Gratian M. Hill T.M. Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.Mol. Genet. Genomics. 2001; 265: 941-953Crossref PubMed Scopus (18) Google Scholar). Residues specifically implicated in this behavior are the G-C(6) base pair of Ter (Figure 1B) and the side chain of Glu49 of Tus. Alternate mechanisms for determination of polarity in this system have been discussed in detail by Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and anti-helicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (103) Google Scholar. The major replicative helicase (DnaB) is the first replisomal protein at a replication fork to encounter the Tus-Ter complex (Schaeffer et al., 2005Schaeffer P.M. Headlam M.J. Dixon N.E. Protein-protein interactions in the eubacterial replisome.IUBMB Life. 2005; 57: 5-12Crossref PubMed Scopus (62) Google Scholar), and binding of Tus has been known for some time to compromise unwinding of Ter DNA by DnaB in vitro in a polar fashion (Lee et al., 1989Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Escherichia coli replication termination protein impedes the action of helicases.Proc. Natl. Acad. Sci. USA. 1989; 86: 9104-9108Crossref PubMed Scopus (112) Google Scholar, Khatri et al., 1989Khatri G.S. MacAllister T. Sista P.R. Bastia D. The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase.Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (124) Google Scholar). A direct physical interaction between Tus and DnaB at the nonpermissive face that prevents unwinding of Ter is a potential mechanism to enforce polarity, and there is experimental support for this (Mulugu et al., 2001Mulugu S. Potnis A. Shamsuzzaman Taylor J. Alexander K. Bastia D. Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction.Proc. Natl. Acad. Sci. USA. 2001; 98: 9569-9574Crossref PubMed Scopus (51) Google Scholar). However, a major difficulty with this mechanism is that the Tus-Ter complex seems to exhibit polarity in inhibition of strand separation by a variety of DNA helicases, including some that move on their single-stranded DNA tracks in the 5′–3′ direction (such as DnaB) and those that have 3′–5′ polarity (e.g., E. coli Rep, PriA, UvrD, and SV40 virus large T antigen) (Lee et al., 1989Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Escherichia coli replication termination protein impedes the action of helicases.Proc. Natl. Acad. Sci. USA. 1989; 86: 9104-9108Crossref PubMed Scopus (112) Google Scholar, Khatri et al., 1989Khatri G.S. MacAllister T. Sista P.R. Bastia D. The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase.Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (124) Google Scholar, Bedrosian and Bastia, 1991Bedrosian C.L. Bastia D. Escherichia coli replication terminator protein impedes simian virus 40 (SV40) DNA replication fork movement and SV40 large tumor antigen helicase activity in vitro at a prokaryotic terminus sequence.Proc. Natl. Acad. Sci. USA. 1991; 88: 2618-2622Crossref PubMed Scopus (28) Google Scholar, Lee and Kornberg, 1992Lee E.H. Kornberg A. Features of replication fork blockage by the Escherichia coli terminus-binding protein.J. Biol. Chem. 1992; 267: 8778-8784Abstract Full Text PDF PubMed Google Scholar, Hidaka et al., 1992Hidaka M. Kobayashi T. Ishimi Y. Seki M. Enomoto T. Abdel-Monem M. Horiuchi T. Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.J. Biol. Chem. 1992; 267: 5361-5365Abstract Full Text PDF PubMed Google Scholar, Sahoo et al., 1995Sahoo T. Mohanty B.K. Lobert M. Manna A.C. Bastia D. The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding.J. Biol. Chem. 1995; 270: 29138-29144Crossref PubMed Scopus (39) Google Scholar). Moreover, transcription by E. coli RNA polymerase is also reported to be blocked in a polar manner (Sahoo et al., 1995Sahoo T. Mohanty B.K. Lobert M. Manna A.C. Bastia D. The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding.J. Biol. Chem. 1995; 270: 29138-29144Crossref PubMed Scopus (39) Google Scholar, Mohanty et al., 1998Mohanty B.K. Sahoo T. Bastia D. Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa.J. Biol. Chem. 1998; 273: 3051-3059Crossref PubMed Scopus (27) Google Scholar). We found previously that the kinetics of interaction of Tus with TerB were affected differently by mutations in Tus, depending on their location at the permissive or the nonpermissive face of the complex, and these data suggested that dissociation of Tus occurs in a stepwise manner (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar). Polarity could then be explained by the existence of a different series of elementary steps in dissociation of the complex when the helicase approaches from one face as opposed to the other (Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and anti-helicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (103) Google Scholar). This led to a more precise definition of this hypothesis, considered here: That approach of DnaB, at the forefront of the replisome, to a Tus-Ter complex engineers a structure in DNA that differentially affects dissociation of Tus depending on the direction of approach. The simplest DNA structure engineered by a helicase is forked duplex DNA, and a simple way to test the hypothesis was to measure the rates of dissociation of Tus from forked variants of TerB that mimic structures that would be produced by helicase action. Here, we first show that the rates of dissociation of Tus from forked TerB oligonucleotides are indeed profoundly different, depending on whether the fork is at one end of TerB or the other. In particular, forks that expose the strictly conserved G-C(6) base pair at the nonpermissive face produce a complex in which Tus is locked onto the DNA: It dissociates about 40-fold more slowly than from wild-type TerB. We trace this locking behavior to a single nucleotide base (C6) of Ter, which we propose forms a new contact with a cryptic cytosine-specific, single-stranded DNA binding site on the surface of Tus. This site is then identified in an X-ray crystal structure of Tus in complex with an appropriate forked duplex version of Ter. Finally, we address the question of what may happen when the later-arriving, oppositely moving replisome approaches the first replisome stalled at the Tus-Ter complex: we show that strand separation at the permissive face can unlock the first complex, displacing Tus to allow replication of the remaining double-stranded DNA at the terminus. These studies thus provide simple and elegant explanations of many of the unresolved questions regarding the mechanism of replication fork arrest at Tus bound Ter sites in the final stages of chromosomal DNA replication. In addition, they reveal an unprecedented stable interaction between a monomeric DNA binding protein and a particular forked DNA structure, which might be exploited in practical ways. The kinetics and thermodynamics of interaction of Tus with TerB and its forked versions were studied by SPR using a Biacore instrument at 20°C in a buffer at pH 7.5 containing 250 mM KCl; 21 nucleotide 5′-biotinylated TerB oligonucleotides were immobilized through an abasic spacer to streptavidin-coated Biacore chip surfaces (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar). Each of the strands of TerB was immobilized separately, and the other (hybridized) strand contained noncomplementary regions (e.g., as shown in Figure 2A). We could thus examine dissociation of Tus from TerB sites containing noncomplementary mutated regions of various lengths on both strands at each end. As observed previously (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar), the interaction of Tus with TerB is a well-behaved interaction for study by SPR. Dissociation generally followed a first-order rate law; half-lives and values of KD for the wild-type complex in both orientations (TerB and rTerB) are given in Figure 2A (complete kinetic and thermodynamic data, including estimates of error limits in all measurements, and sequences of these and all other oligonucleotides are given in Figure S1 in the Supplemental Data available with this article online). The data for TerB and rTerB indicate that the orientation of the wild-type duplex with respect to the surface has little effect on binding parameters. Values of KD were 1–2 nM; use of 250 mM KCl in the buffer brings parameters into a range reliably quantifiable by SPR (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar). As the forked region was progressively extended at the permissive end of TerB, dissociation rates became progressively faster, and it mattered little which strand was mutated (Figures 2A and 2B) or if either of them was removed completely (Figure S1). It is clear that strand separation even as far as A-T(20) of TerB would lead to rapid dissociation of Tus (Figure 2A, oligonucleotides F3p-TerB and F3p-rTerB), resulting in unimpeded progression of the replisome through Ter (Figure 2C). The data are thus consistent with removal of Tus due to the progressive loss of protein-DNA contacts during strand separation by the helicase at the permissive end of Ter. The situation was different with single-stranded regions at the nonpermissive end. A 5- to 7-fold increase in KD was observed when the mismatched regions were three or four nucleotides long, with dissociation rates similar to those with wild-type TerB regardless of which strand was mutated (Figures 3A and 3B). However, strand specificity became dramatically obvious when the forked region extended to the G-C(6) base pair. When the strand containing C(6) was mutated (the bottom strand in Figure 3A; oligonucleotide F5n-TerB), Tus dissociated about twice as rapidly as from TerB (Figures 3A and 3C), and KD increased almost 30-fold. On the other hand, mutation of the top strand (F5n-rTerB) resulted in Tus being firmly locked onto the forked TerB (Figures 3A–3C): Tus dissociated about 40-fold more slowly than from TerB, and KD was about 3-fold lower. Although extension of the fork to include T-A(7) (F6n-rTerB) resulted in a similarly locked behavior, its further lengthening to A-T(8) (F7n-rTerB) resulted in poorer binding because of a slower association rate (Figures 3A and S1). It is clear therefore that strand separation by a helicase approaching from the nonpermissive face of the Tus-Ter complex would lead to a locked complex that is even more stable than the regular complex with fully duplex TerB, while at the permissive face helicase action would simply promote dissociation of Tus. These observations provide an adequate explanation of the polarity observed in replication termination. It is clear that the strictly conserved C(6) base on the bottom strand of the TerB sequence in Figure 3A must not be base-paired to lock the complex. Some further experiments verified this. The locking behavior still occurred when the first five residues of the mutant strand in F5n-rTerB were completely removed (Δ5n-rTerB; Figure 3A), indicating that a forked structure is not required and systematic mutagenesis of each of the first five residues of the wild-type strand of F5n-rTerB showed that mutagenesis of C(6), and only C(6), abrogated the locking behavior of the complex (Figures 3A, 3C, and 3D). Indeed, complete removal of the first four residues on the 3′ strand, leaving only C(6), still resulted in formation of a locked species (“single O/H C,” Figure 3A). The unpaired C(6) residue is thus necessary and sufficient for lock formation. The T-A(7) base pair in TerB is not conserved, being replaced by G-C in TerC and two other Ter sites (Figure 1B). Its mutagenesis resulted in only small effects on the strength of the Tus-TerB interaction or in vivo fork arrest activity (Coskun-Ari and Hill, 1997Coskun-Ari F.F. Hill T.M. Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.J. Biol. Chem. 1997; 272: 26448-26456Crossref PubMed Scopus (47) Google Scholar). Data in Figure 3E show that changing T-A(7) to G-C has very little effect on formation or stability of the locked species and that substitution of C(6) by G again results in loss of the locking behavior. These data indicate that a molecular mousetrap operates during replication fork arrest at the nonpermissive face of Tus-Ter (Figure 3F). The trap is set by the binding of Tus to the Ter site, and it is sprung by strand separation by DnaB at the forefront of the approaching replisome. This results in the flipping of the C(6) residue out of the double helix by rotation of the phosphodiester backbone and its base-specific binding in a cryptic cytosine-specific binding pocket in or near the DNA binding channel of Tus. Other contacts of Tus with the displaced strand may occur, but they are not sequence specific. Base-flipping processes that bear some similarity to this occur in DNA modification and repair enzymes like DNA N-glycosylases, apurinic endonucleases, and DNA methyltransferases (Cheng and Roberts, 2001Cheng X. Roberts R.J. AdoMet-dependent methylation, DNA methyltransferases and base flipping.Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (380) Google Scholar). This mechanism explains the observation that mutagenesis of the G-C(6) base pair of TerB compromises fork arrest without severely affecting Tus binding (Coskun-Ari and Hill, 1997Coskun-Ari F.F. Hill T.M. Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.J. Biol. Chem. 1997; 272: 26448-26456Crossref PubMed Scopus (47) Google Scholar) and the strict conservation of this base pair in all Ter sites (Figure 1B). Specific physical interaction of DnaB with Tus is not precluded but would appear to be unnecessary. Several further experiments were carried out to study aspects of this model. We anticipated that Tus would dissociate very slowly from oligonucleotides that expose C(6) at the nonpermissive end in 200 mM potassium glutamate buffer at 25°C, conditions that have been used in many earlier studies of the Tus-Ter interaction (e.g., see Skokotas et al., 1995Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.J. Biol. Chem. 1995; 270: 30941-30948Crossref PubMed Scopus (13) Google Scholar, Henderson et al., 2001Henderson T.A. Nilles A.F. Valjavec-Gratian M. Hill T.M. Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.Mol. Genet. Genomics. 2001; 265: 941-953Crossref PubMed Scopus (18) Google Scholar); the half-life of the wild-type Tus-TerB complex is 150 min in this buffer. Accordingly, measurements of dissociation of Tus from 32P-labeled TerB and partial-duplex TerB derivatives (Table 1) in solution were made using the conventional filter binding assay. Complexes with Tus were challenged with excess unlabeled TerB oligonucleotide, and samples were filtered at various times to determine the proportion of protein bound 32P remaining. Dissociation of Tus generally followed a first-order rate law; half-lives of the complexes are given in Table 1. It is apparent that dissociation half-lives in glutamate buffer were much more similar for the wild-type TerB oligonucleotide and those that expose C(6), indicating that the locked conformation of the DNA either no longer forms under these conditions or, more likely, that its dissociation from Tus occurs at a similar rate as from wild-type TerB, i.e., existence of the lock is masked by the higher stability of the wild-type complex.Table 1Half-lives for Dissociation of Tus-Ter Complexes in 200 mM Potassium GlutamateOligonucleotideaThe core TerB sequences are overlined.Half-life (min)bAverage of three independent experiments (± SEM), using a competition filter binding assay in KG200 buffer at 25°C (Skokotas et al., 1995).150 ± 6131 ± 7205 ± 8a The core TerB sequences are overlined.b Average of three independent experiments (± SEM), using a competition filter binding assay in KG200 buffer at 25°C (Skokotas et al., 1995Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.J. Biol. Chem. 1995; 270: 30941-30948Crossref PubMed Scopus (13) Google Scholar). Open table in a new tab These observations prompted examination of the effects of ionic strength on dissociation rate constants (kd) as measured by SPR (Figure 3G). At high ionic strength, a large difference in kd was observed for F5n-rTerB cf. rTerB, with little dependence on ionic strength. At low ionic strength, the two lines in Figure 3G have a steeper slope and converge. The slopes of lines in such log/log plots are directly related to the numbers of ionic contacts that need to be disrupted during the rate-determining step in the dissociation of a protein from a DNA complex (Record et al., 1991Record Jr., M.T. Ha J.-H. Fisher M.A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA.Methods Enzymol. 1991; 208: 291-343Crossref PubMed Scopus (274) Google Scholar). These data therefore offer further support for a stepwise mechanism for dissociation of Tus from both TerB (Neylon et al., 2000Neylon C. Brown S.E. Kralicek A.V. Miles C.S. Love C.A. Dixon N.E. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.Biochemistry. 2000; 39: 11989-11999Crossref PubMed Scopus (126) Google Scholar) and the forked species, and they show that the rate-determining step in each process changes abruptly with ionic strength. With both oligonucleotides, the slowest step in dissociation at high ionic strength involves loss of a single (or few) ionic interaction(s), while at low ionic strength the rate-determining step requires disruption of a much larger number of such interactions. The slowest step in dissociation of Tus from the locked complex at higher salt concentrations is likely to be removal of the C(6) base from its binding pocket, while for the wild-type complex, it is the breakage of a particular but undetermined site-specific interaction. At a physiological ionic strength corresponding to 150 mM KCl, the half-lives for the wild-type and locked complexes were still very different, being about 80 and 490 min, respectively (Figure 3G). Thus, the more stable locked species would be expected to be generated by the action of DnaB under cellular conditions. Duggan et al., 1996Duggan L.J. Asmann P.T. Hill T.M. Gottlieb P.A. Identification of a Tus protein segment that photo-cross-links with TerB DNA and elucidation of the role of certain thymine methyl groups in the Tus-TerB complex using halogenated uracil analogues.Biochemistry. 1996; 35:" @default.
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• W2046332670 title "A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli" @default.
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