title: Hexapeptides that inhibit processing of branched DNA structures induce a dynamic ensemble of Holliday junction conformations.
abstract: 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 Mg(2+) 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.
authors.affiliation: From the Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712.
authors.forename: Brian
authors.initials: B
authors.lastname: Cannon
authors.affiliation: From the Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712.
authors.forename: Aashiq H
authors.initials: AH
authors.lastname: Kachroo
authors.affiliation: From the Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712.
authors.forename: Inga
authors.initials: I
authors.lastname: Jarmoskaite
authors.affiliation: From the Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712 jayaram@austin.utexas.edu.
authors.forename: Makkuni
authors.initials: M
authors.lastname: Jayaram
authors.affiliation: From the Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712 rick_russell@cm.utexas.edu.
authors.forename: Rick
authors.initials: R
authors.lastname: Russell
mesh_terms: D045566:DNA, Cruciform / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D009690:Nucleic Acid Conformation; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D045566:DNA, Cruciform; D009842:Oligopeptides
keywords: DNA; DNA recombination; Holliday junction; fluorescence resonance energy transfer (FRET); gel mobility shift; single-molecule biophysics; smFRET; small angle x-ray scattering