title: Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.
abstract: The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg(2+), low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg(2+). 4WJ-PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2), while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (K(D)s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.
authors: Department of Chemistry, New York University, New York, New York.
authors: Filbert
authors: F
authors: Totsingan
authors: Anthony J
authors: AJ
authors: Bell
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D024243:HMGB1 Protein / Q000737:chemistry* / Q000378:metabolism*; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; C104200:Hbp1 protein, rat; D006657:Histones; D020135:Peptide Nucleic Acids; D011994:Recombinant Proteins
keywords: circular dichroism; cruciform DNA; four-way junction; high mobility group protein B 1 (HMGB1); histone H1; peptide nucleic acid (PNA)
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