title: HU binding to a DNA four-way junction probed by Förster resonance energy transfer.
abstract: The Escherichia coli protein HU is a non-sequence-specific DNA-binding protein that interacts with DNA primarily through electrostatic interactions. In addition to nonspecific binding to linear DNA, HU has been shown to bind with nanomolar affinity to discontinuous DNA substrates, such as repair and recombination intermediates. This work specifically examines the HU-four-way junction (4WJ) interaction using fluorescence spectroscopic methods. The conformation of the junction in the presence of different counterions was investigated by Förster resonance energy transfer (FRET) measurements, which revealed an ion-type conformational dependence, where Na(+) yields the most stacked conformation followed by K(+) and Mg(2+). HU binding induces a greater degree of stacking in the Na(+)-stabilized and Mg(2+)-stabilized junctions but not the K(+)-stabilized junction, which is attributed to differences in the size of the ionic radii and potential differences in ion binding sites. Interestingly, junction conformation modulates binding affinity, where HU exhibits the lowest affinity for the Mg(2+)-stabilized form (24 μM(-1)), which is the least stacked conformation. Protein binding to a mixed population of open and stacked forms of the junction leads to nearly complete formation of a protein-stabilized stacked-X junction. These results strongly support a model in which HU binds to and stabilizes the stacked-X conformation.
authors.affiliation: Molecular Biology and Biochemistry Department, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459-0175, United States.
authors.forename: Codruta Iulia
authors.initials: CI
authors.lastname: Vitoc
authors.forename: Ishita
authors.initials: I
authors.lastname: Mukerji
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D045566:DNA, Cruciform / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015964:Gene Expression Regulation, Bacterial; D008274:Magnesium / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C116822:hns protein, E coli; D012964:Sodium; D008274:Magnesium; D011188:Potassium