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Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations*

  • Brian Cannon
    Footnotes
    Affiliations
    Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
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  • Aashiq H. Kachroo
    Footnotes
    Affiliations
    Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
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  • Inga Jarmoskaite
    Footnotes
    Affiliations
    Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
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  • Makkuni Jayaram
    Correspondence
    To whom correspondence may be addressed: Dept. of Molecular Biosciences and Inst. for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Tel.: 512-471-0966; Fax: 512-471-1218.
    Affiliations
    Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
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  • Rick Russell
    Correspondence
    To whom correspondence may be addressed: Dept. of Molecular Biosciences and Inst. for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Tel.: 512-471-1514; Fax: 512-232-3432.
    Affiliations
    Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant GM070456 (to R. R.), Robert F. Welch Foundation Grants F-1563 (to R. R.) and F-1274 (to M. J.), and National Science Foundation Grant MCB1049925 (to M. J.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 These authors contributed equally to this work.
    2 Present address: Dept. of Physics, Loyola University Chicago, Chicago, IL 60660.
    3 Present address: Dept. of Biochemistry, Stanford University, Stanford, CA 94305.
Open AccessPublished:July 24, 2015DOI:https://doi.org/10.1074/jbc.M115.663930
      Holliday junctions are critical intermediates in DNA recombination, repair, and restart of blocked replication. Hexapeptides have been identified that bind to junctions and inhibit various junction-processing enzymes, and these peptides confer anti-microbial and anti-tumor properties. Earlier studies suggested that inhibition results from stabilization of peptide-bound Holliday junctions in the square planar conformation. Here, we use single molecule fluorescence resonance energy transfer (smFRET) and two model junctions, which are AT- or GC-rich at the branch points, to show that binding of the peptide KWWCRW induces a dynamic ensemble of junction conformations that differs from both the square planar and stacked X conformations. The specific features of the conformational distributions differ for the two peptide-bound junctions, but both junctions display greatly decreased Mg2+ dependence and increased conformational fluctuations. The smFRET results, complemented by gel mobility shift and small angle x-ray scattering analyses, reveal structural effects of peptides and highlight the sensitivity of smFRET for analyzing complex mixtures of DNA structures. The peptide-induced conformational dynamics suggest multiple stacking arrangements of aromatic amino acids with the nucleobases at the junction core. This conformational heterogeneity may inhibit DNA processing by increasing the population of inactive junction conformations, thereby preventing the binding of processing enzymes and/or resulting in their premature dissociation.

      Introduction

      The Holliday junction is a four-way branched DNA structure that is a key intermediate in fundamental biological processes including homologous recombination, DNA damage repair, and restart of blocked replication forks (
      • Holliday R.
      A mechanism for gene conversion in fungi.
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      • Whitby M.C.
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      • Lloyd R.G.
      Reverse branch migration of Holliday junctions by RecG protein: a new mechanism for resolution of intermediates in recombination and DNA repair.
      ,
      • Kowalczykowski S.C.
      Initiation of genetic recombination and recombination-dependent replication.
      ,
      • Maher R.L.
      • Branagan A.M.
      • Morrical S.W.
      Coordination of DNA replication and recombination activities in the maintenance of genome stability.
      ,
      • Sarbajna S.
      • West S.C.
      Holliday junction processing enzymes as guardians of genome stability.
      ). Holliday junctions are also produced during DNA exchange mediated by conservative site-specific recombinases of the tyrosine family (
      • Grindley N.D.
      • Whiteson K.L.
      • Rice P.A.
      Mechanisms of site-specific recombination.
      ,
      • Jayaram M.
      • Ma C.H.
      • Kachroo A.H.
      • Rowley P.A.
      • Guga P.
      • Fan H.F.
      • Voziyanov Y.
      An overview of tyrosine recombinases: from a Flp perspective.
      ,
      • Van Duyne G.D.
      Cre recombinase.
      ). In recombination by these enzymes, cleavage and exchange of the first pair of strands result in a Holliday junction intermediate, which then isomerizes to trigger the second strand cleavage exchange step, resolving the junction into recombinant products.
      The conformations of Holliday junctions are exquisitely sensitive to their ionic environments. At low ionic strength, repulsion of the negative charges at the core and along the DNA backbone causes the junction to adopt a nearly square planar form, with the extended arms arranged in a cruciform-like shape (
      • Duckett D.R.
      • Murchie A.I.
      • Diekmann S.
      • von Kitzing E.
      • Kemper B.
      • Lilley D.M.
      The structure of the Holliday junction, and its resolution.
      ,
      • Clegg R.M.
      • Murchie A.I.
      • Lilley D.M.
      The solution structure of the four-way DNA junction at low-salt conditions: a fluorescence resonance energy transfer analysis.
      ). In high monovalent ion concentrations or modest concentrations of divalent cations such as Mg2+, the arms stack co-axially to form an anti-parallel right-handed X-shaped structure (
      • Duckett D.R.
      • Murchie A.I.
      • Diekmann S.
      • von Kitzing E.
      • Kemper B.
      • Lilley D.M.
      The structure of the Holliday junction, and its resolution.
      ,
      • Churchill M.E.
      • Tullius T.D.
      • Kallenbach N.R.
      • Seeman N.C.
      A Holliday recombination intermediate is twofold symmetric.
      ,
      • Murchie A.I.
      • Clegg R.M.
      • von Kitzing E.
      • Duckett D.R.
      • Diekmann S.
      • Lilley D.M.
      Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.
      ,
      • Clegg R.M.
      • Murchie A.I.
      • Zechel A.
      • Carlberg C.
      • Diekmann S.
      • Lilley D.M.
      Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.
      ). Each arm may stack with either of two partners, generating two possible conformations between which the junction may oscillate (
      • Murchie A.I.
      • Portugal J.
      • Lilley D.M.
      Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange.
      ,
      • Grainger R.J.
      • Murchie A.I.
      • Lilley D.M.
      Exchange between stacking conformers in a four-Way DNA junction.
      ) (see Fig. 1A). The relative abundance of these conformations is determined by sequence-specific interactions at the branch point and is unaffected by the Mg2+ concentration (
      • McKinney S.A.
      • Déclais A.C.
      • Lilley D.M.
      • Ha T.
      Structural dynamics of individual Holliday junctions.
      ,
      • Liu J.
      • Declais A.C.
      • McKinney S.A.
      • Ha T.
      • Norman D.G.
      • Lilley D.M.
      Stereospecific effects determine the structure of a four-way DNA junction.
      ). The transition rates between the conformations decrease with increasing Mg2+, most likely because transit through obligatory square planar or tetrahedral intermediates depends on a loss of Mg2+ ions from the junction (
      • McKinney S.A.
      • Déclais A.C.
      • Lilley D.M.
      • Ha T.
      Structural dynamics of individual Holliday junctions.
      ,
      • Joo C.
      • McKinney S.A.
      • Lilley D.M.
      • Ha T.
      Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy.
      ,
      • Yu J.
      • Ha T.
      • Schulten K.
      Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.
      ,
      • Hohng S.
      • Zhou R.
      • Nahas M.K.
      • Yu J.
      • Schulten K.
      • Lilley D.M.
      • Ha T.
      Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction.
      ). Although the canonical stacked X and square planar conformations have provided a very useful framework for understanding junction structures and dynamics, recent studies have shown that a broader range of conformations is accessible as transient intermediates or even stable states in isolation or when bound by a protein (
      • Yu J.
      • Ha T.
      • Schulten K.
      Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.
      ,
      • Hohng S.
      • Zhou R.
      • Nahas M.K.
      • Yu J.
      • Schulten K.
      • Lilley D.M.
      • Ha T.
      Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction.
      ,
      • Biertümpfel C.
      • Yang W.
      • Suck D.
      Crystal structure of T4 endonuclease VII resolving a Holliday junction.
      ,
      • Karymov M.A.
      • Chinnaraj M.
      • Bogdanov A.
      • Srinivasan A.R.
      • Zheng G.
      • Olson W.K.
      • Lyubchenko Y.L.
      Structure, dynamics, and branch migration of a DNA Holliday junction: a single-molecule fluorescence and modeling study.
      ) (see “Discussion”).
      Figure thumbnail gr1
      FIGURE 1Holliday junctions used in the smFRET experiments. A, the four 11-bp arms of the junctions (termed B, H, R, and X). The 5′ ends of the B and H arms were labeled with Cy5 (red circle) and Cy3 (green circle) dyes, respectively. The R arm included a 5′ biotin (brown circle) for immobilizing the junctions to streptavidin-coated slides. The junction can fold into two possible anti-parallel conformations, IsoI and IsoII, which give distinct FRET signals because of the relative positions of the dyes. B, the nucleotide sequences surrounding the immobile branch point for the AT and GC junctions are shown. The two junctions are identical in the rest of the arm sequences, which are not shown.
      In principle, molecules that interfere with or destabilize the functional conformations of Holliday junctions can be deployed to inhibit biological processes that generate these junctions as reaction intermediates. Such molecules could potentially serve as effective anti-microbial agents. Indeed, through screens of a combinatorial library, short synthetic hexapeptides with these properties have been identified (
      • Cassell G.
      • Klemm M.
      • Pinilla C.
      • Segall A.
      Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.
      ,
      • Boldt J.L.
      • Pinilla C.
      • Segall A.M.
      Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.
      ,
      • Gunderson C.W.
      • Segall A.M.
      DNA repair, a novel antibacterial target: Holliday junction-trapping peptides induce DNA damage and chromosome segregation defects.
      ,
      • Su L.Y.
      • Willner D.L.
      • Segall A.M.
      An antimicrobial peptide that targets DNA repair intermediates in vitro inhibits Salmonella growth within murine macrophages.
      ). These peptides have been shown to impede the unwinding of branched DNA substrates by the RecG helicase of Escherichia coli, to interfere with Holliday junction resolution by the RuvABC complex, and to inhibit site-specific recombination by tyrosine recombinases, with accumulation of the Holliday junction intermediate (
      • Cassell G.
      • Klemm M.
      • Pinilla C.
      • Segall A.
      Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.
      ,
      • Boldt J.L.
      • Pinilla C.
      • Segall A.M.
      Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.
      ,
      • Klemm M.
      • Cheng C.
      • Cassell G.
      • Shuman S.
      • Segall A.M.
      Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.
      ,
      • Cassell G.D.
      • Segall A.M.
      Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange.
      ,
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Rajeev L.
      • Segall A.
      • Gardner J.
      The bacteroides NBU1 integrase performs a homology-independent strand exchange to form a holliday junction intermediate.
      ,
      • Gunderson C.W.
      • Boldt J.L.
      • Authement R.N.
      • Segall A.M.
      Peptide wrwycr inhibits the excision of several prophages and traps holliday junctions inside bacteria.
      ).
      Previous studies have used gel mobility shift, fluorescence, and x-ray crystallography to probe the interactions of the inhibitory peptides with synthetic Holliday junctions (
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ,
      • Rideout M.C.
      • Naili I.
      • Boldt J.L.
      • Flores-Fujimoto A.
      • Patra S.
      • Rostron J.E.
      • Segall A.M.
      wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.
      ). Although these studies have not revealed a discrete conformation for a bound peptide, the data support a model in which a peptide dimer, typically mediated by a disulfide linkage between cysteines, binds within the central core of the junction and stabilizes it in a square planar conformation (
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ). The peptides are rich in aromatic amino acids and are thought to derive binding energy from stacking interactions with solvent-exposed bases, stabilizing the square planar conformation (
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ). However, if the only effect of peptide binding were to stabilize this conformation, peptide binding would most simply be expected to activate its target enzymes rather than inhibit them because these enzymes bind preferentially to square planar conformations (
      • Whitby M.C.
      • Lloyd R.G.
      Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli.
      ,
      • Lee J.
      • Voziyanov Y.
      • Pathania S.
      • Jayaram M.
      Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase.
      ,
      • Hargreaves D.
      • Rice D.W.
      • Sedelnikova S.E.
      • Artymiuk P.J.
      • Lloyd R.G.
      • Rafferty J.B.
      Crystal structure of E. coli RuvA with bound DNA Holliday junction at 6 Å resolution.
      ,
      • Sharples G.J.
      • Ingleston S.M.
      • Lloyd R.G.
      Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA.
      ,
      • Gopaul D.N.
      • Guo F.
      • Van Duyne G.D.
      Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Chen Y.
      • Narendra U.
      • Iype L.E.
      • Cox M.M.
      • Rice P.A.
      Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping.
      ,
      • Fogg J.M.
      • Kvaratskhelia M.
      • White M.F.
      • Lilley D.M.
      Distortion of DNA junctions imposed by the binding of resolving enzymes: a fluorescence study.
      ). Thus, instead of inhibition arising from changes to the global properties of the junctions, the peptides have been suggested to inhibit various enzymes by binding DNA junctions competitively or by binding to an enzyme-substrate complex and producing local changes at the active sites that block enzyme activity (
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ). Although some peptides can inhibit the initial cleavage step at high concentrations, the dominant effect is to stall recombination after the first cleavage exchange step by trapping the Holliday junction, underscoring the role of junction binding by these peptides in their inhibition of recombination reactions.
      To further probe the structural effects of inhibitory hexapeptides on Holliday junctions, here we focus on the interactions of one of the most potent peptides, KWWCRW (
      • Klemm M.
      • Cheng C.
      • Cassell G.
      • Shuman S.
      • Segall A.M.
      Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.
      ,
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ), with two Holliday junctions that differ in the sequences surrounding their branch points (AT-rich and GC-rich; see Fig. 1B). We perform single molecule fluorescence resonance energy transfer (smFRET)
      The abbreviations used are: smFRET
      single molecule FRET
      SAXS
      small angle x-ray scattering
      TD
      transition density.
      analysis, along with native gel shift and small angle x-ray scattering (SAXS) assays, of the peptide-free and peptide-bound junctions. All three methods indicate that peptide binding changes the conformations of the DNA arms. The gel mobilities of the peptide-bound junctions do not conform to canonical square planar or stacked X conformations, whereas the SAXS data suggest junction conformations that are more open than stacked X conformations. The higher resolution of smFRET reveals additional detail, indicating an ensemble of conformations that fluctuate rapidly, with substantial effects of junction sequence on the dominant conformations. Together, the findings from the three approaches suggest a potential mechanism for peptide action in which inhibition of junction processing results from a decreased population of the biologically active state of the junction and a concomitant increase in multiple intermediates that are in rapid dynamic exchange.

      Discussion

      The mechanisms by which anti-microbial hexapeptides interact with three- and four-way DNA junctions and inhibit DNA unwinding, branch migration, or junction resolution are not well understood. Based on gel mobility shift, fluorescence quenching, and structural data, combined with modeling, it has been suggested that an inhibitory peptide dimer competes with processing enzymes for binding branched DNA structures, accelerates the dissociation of bound enzymes, and/or induces a ternary complex conformation in which catalysis is deterred (
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ,
      • Rideout M.C.
      • Naili I.
      • Boldt J.L.
      • Flores-Fujimoto A.
      • Patra S.
      • Rostron J.E.
      • Segall A.M.
      wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.
      ). Here, we used smFRET, gel mobility shift assay, and SAXS to probe the effects of one of these peptides on the global conformations of two Holliday junctions. Our results indicate an additional, unrecognized effect of the peptide. We find that peptide binding increases the range and dynamics of junction conformations and greatly reduces their dependence on interactions with Mg2+ ion. The formation of these rapidly exchanging conformations upon peptide binding, which by definition will include conformations that are not productive for enzyme binding or function, may be a key factor in the inhibition of Holliday junction processing enzymes.

      Peptide Binding to the Holliday Junction Core Generates Conformational Diversity and a Flattening of the Energy Landscape

      The previously uncharacterized AT junction follows the general behavior of other synthetic junctions in assuming the square planar conformation in the absence of Mg2+ and the IsoI and IsoII stacked X conformations in its presence. As observed for the GC junction (
      • McKinney S.A.
      • Déclais A.C.
      • Lilley D.M.
      • Ha T.
      Structural dynamics of individual Holliday junctions.
      ), the rate constants for transitions of the AT junction between IsoI and IsoII decrease with increased Mg2+ concentration with no change in the equilibrium value, reflecting a requirement for transient unstacking of the bases at the junction core during exchange of the two conformations. The detectable preference for IsoII must arise from sequence differences relative to the GC junction, which populates IsoI and IsoII equally, but the specific origins of the differences are not clear. Previous work has shown that conformer preferences can be influenced by the branch point nucleotides (
      • Grainger R.J.
      • Murchie A.I.
      • Lilley D.M.
      Exchange between stacking conformers in a four-Way DNA junction.
      ,
      • McKinney S.A.
      • Déclais A.C.
      • Lilley D.M.
      • Ha T.
      Structural dynamics of individual Holliday junctions.
      ), their nearest neighbors (
      • Miick S.M.
      • Fee R.S.
      • Millar D.P.
      • Chazin W.J.
      Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions.
      ), and even the third base pairs from the junction (
      • Grainger R.J.
      • Murchie A.I.
      • Lilley D.M.
      Exchange between stacking conformers in a four-Way DNA junction.
      ), all of which include differences between the AT and GC junctions.
      Upon binding KWWCRW, the conformational properties of both junctions are altered dramatically. In the absence of Mg2+, both junctions display FRET histograms with increased average values and broadened distributions, indicating the formation of conformational ensembles distinct from the square planar form. With bound peptide, the FRET distributions of both junctions are relatively insensitive to Mg2+ and have average values between those of the two stacked X conformations. The simplest interpretation of these results is that the conformational ensembles also include open conformations, distinct from the stacked X conformations. The TD and σFRET analyses show that the broadening of the FRET distributions is present on a molecule by molecule basis, particularly for the AT junction, indicating dynamic transitions between conformations.
      We considered whether the changes in FRET behavior might arise from an artifactual effect of the peptide on one or both dyes rather than an effect on the junction conformations, but we rejected this hypothesis because: (i) the AT and GC junctions respond differently to the presence of peptide, whereas an effect on the dyes would be expected to be the same; (ii) the junction behaviors persisted for minutes after the peptide was washed out in dissociation experiments, ruling out the effect requiring high peptide concentrations as might be expected for a weak, nonspecific interaction with the dyes; (iii) the behaviors of the junctions ultimately returned to their intrinsic behaviors after peptide washout, ruling out irreversible effects; and (iv) the total intensity distribution (Cy3 + Cy5) was unchanged by the peptide (data not shown), ruling out quenching by the peptide.
      Although an alternative interpretation of the smFRET results would be that the junctions transition between stacked X conformations fast enough that they cannot be resolved in our measurements, this interpretation is inconsistent with the SAXS results, which show that peptide binding increases the population of open conformations at low Mg2+ concentration. The gel mobility shifts are also consistent with the FRET data, indicating nonstandard junction conformations spawned by peptide binding, without conveying the diversity of these conformations or fluctuations within them.
      At a structural level, the effect of the peptides most likely arises because the positively charged amino acids promote DNA binding, reducing the dependence on Mg2+ concentration, and the hydrophobic amino acids can stack in various arrangements with the nucleobases at the junction core (
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ,
      • Rideout M.C.
      • Naili I.
      • Boldt J.L.
      • Flores-Fujimoto A.
      • Patra S.
      • Rostron J.E.
      • Segall A.M.
      wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.
      ), resulting in an array of conformations with various angles between the helical arms. In the parlance of an energy landscape representing the conformational space available to the junction, the peptide induces landscape flattening such that multiple conformations are approximately equivalent in free energy and therefore populated. As suggested by the SAXS analysis, the peptide-induced ensembles are dominated by conformations that are more open than the stacked X conformations. Nevertheless, the peak FRET values are distinct from those observed under the low ionic strength conditions that favor the square planar conformations, indicating a set of distinct conformations. Although the canonical model involving stacked X and square planar conformations has provided an important foundation for understanding junction conformations and dynamics, there is increasing evidence for additional, noncanonical conformations along the pathways for exchange between stacked X conformations (
      • Yu J.
      • Ha T.
      • Schulten K.
      Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.
      ,
      • Hohng S.
      • Zhou R.
      • Nahas M.K.
      • Yu J.
      • Schulten K.
      • Lilley D.M.
      • Ha T.
      Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction.
      ), as stable ground states for mobile junctions (
      • Karymov M.A.
      • Chinnaraj M.
      • Bogdanov A.
      • Srinivasan A.R.
      • Zheng G.
      • Olson W.K.
      • Lyubchenko Y.L.
      Structure, dynamics, and branch migration of a DNA Holliday junction: a single-molecule fluorescence and modeling study.
      ), or in complexes with junction-binding proteins (
      • Biertümpfel C.
      • Yang W.
      • Suck D.
      Crystal structure of T4 endonuclease VII resolving a Holliday junction.
      ). It is possible that the peptide-induced conformational ensembles observed here resemble one or more of these noncanonical junction conformations.
      In addition to equalizing the valley depths, the peptide decreases the peak barriers between them, facilitating conformational exchanges. This effect is most obvious with high Mg2+ concentrations, where junction transitions in the absence of peptide are slow enough to be measured by smFRET. The rapid structural transitions coupled with the long lifetime for peptide binding to the junction imply that the patterns of stacking interactions between the amino acids and the nucleobases at the junction can fluctuate readily and repeatedly while the peptide remains bound to the junction.

      Differences in Peptide Effects on the AT and GC Junctions

      Although the two junctions share some properties upon binding peptide, there are also notable differences. Most prominently, the GC junction occupies a more closely nested set of conformations, with FRET values that more closely resemble the high FRET IsoII than the IsoI or square planar forms. Nevertheless, it is possible that the same underlying physical model accounts for the peptide effects on the two junctions. For the GC junction, peptide binding may similarly induce conformations that include stacking between amino acid side chains and the junction nucleobases, replacing base-base stacking, but with an energetic preference for conformations with helical arm arrangements similar to the IsoII conformation. Even a modest energetic preference of 1–2 kcal/mol would produce the observed narrowing of the FRET distribution.

      Inhibition of Enzyme Activity

      Hexapeptides such as KWWCRW have been shown to inhibit a broad group of enzymes that catalyze reactions involving branched DNA structures as substrates or intermediates (
      • Boldt J.L.
      • Pinilla C.
      • Segall A.M.
      Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.
      ,
      • Klemm M.
      • Cheng C.
      • Cassell G.
      • Shuman S.
      • Segall A.M.
      Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.
      ,
      • Cassell G.D.
      • Segall A.M.
      Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange.
      ,
      • Kepple K.V.
      • Boldt J.L.
      • Segall A.M.
      Holliday junction-binding peptides inhibit distinct junction-processing enzymes.
      ,
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ,
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ). Although the mechanisms of inhibition have not been established and may vary among proteins, there are several reasons to think that the ability of bound peptide to shift the conformational distribution away from square planar and increase dynamics may contribute to enzyme inhibition.
      First, a common theme of the diverse group of inhibited enzymes is that they act on junctions in a square planar-like conformation, suggesting that a shift away from this conformation could result in inhibition. Second, peptide binding has been shown to accelerate dissociation of at least one protein (RecG), ruling out a model involving strictly competitive inhibition in which the peptide simply occupies the same site as the protein (
      • Kepple K.V.
      • Patel N.
      • Salamon P.
      • Segall A.M.
      Interactions between branched DNAs and peptide inhibitors of DNA repair.
      ). In addition, x-ray crystallography shows a peptide ternary complex with a recombinase-bound DNA junction in which the peptide is bound away from the protein-binding site at the center of the junction (
      • Ghosh K.
      • Lau C.K.
      • Guo F.
      • Segall A.M.
      • Van Duyne G.D.
      Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination.
      ), consistent with global rather than local effects. Finally, the observations of inhibition of multiple enzymes by multiple peptides suggest a generalized mode of action such as that observed here. Peptide-induced effects on the conformational ensemble and dynamics of junctions can be imagined to accelerate protein dissociation, slow binding, and/or inhibit catalytic steps by increasing the population of nonproductive conformations. Although there may also be sources of inhibition that are local and specific to certain enzymes and junctions, it seems likely that the peptide-induced increases in junction conformations and dynamics observed here are linked to inhibition. If the increased range of conformations is important, a simple prediction is that junctions with sequences corresponding to the AT junction will be more strongly inhibited than those corresponding to the GC junction.

      Conclusions

      The application of smFRET to synthetically assembled nucleic acid structures that model their biologically relevant counterparts is uniquely suited for exploring the role of ligand-mediated conformational dynamics and/or diversity in DNA transactions. Our results demonstrate that a hexapeptide broadens the conformational distribution of DNA junctions. The principle of blocking DNA transactions by enhancing, rather than constraining conformational freedom, may be more broadly applicable to a variety of peptide and non-peptide ligands that recognize specific DNA structures. A subset of such agents may engender desirable anti-microbial activities.

      Author Contributions

      A. H. K., B. C., M. J., and R. R. designed the study. A. H. K. and B. C. prepared the junction molecules. B. C. performed and analyzed the smFRET experiments. A. H. K. and B. C. performed the gel shift experiments. I. J. performed the SAXS experiments. B. C., A. H. K., M. J., and R. R. wrote the manuscript. All authors analyzed results and approved the final version of the manuscript.

      Acknowledgments

      We thank Soenke Seifert for assistance on Beamline 12-ID-C.

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