Competition between HMG-I(Y), HMG-1 and histone H1 on four-way junction DNA.
High mobility group proteins HMG-I(Y) and HMG-1, as well as histone H1, all share the common property of binding to four-way junction DNA (4H), a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events. The structure of 4H has also been hypothesized to mimic the DNA crossovers occurring at, or near, the entrance and exit sites on the nucleosome. Furthermore, upon binding to either duplex DNA or chromatin, all three of these nuclear proteins share the ability to significantly alter the structure of bound substrates. In order to further elucidate their substrate binding abilities, electrophoretic mobility shift assays were employed to investigate the relative binding capabilities of HMG-I(Y), HMG-1 and H1 to 4H in vitro. Data indicate a definite hierarchy of binding preference by these proteins for 4H, with HMG-I(Y) having the highest affinity (Kd approximately 6.5 nM) when compared with either H1 (Kd approximately 16 nM) or HMG-1 (Kd approximately 80 nM). Competition/titration assays demonstrated that all three proteins bind most tightly to the same site on 4H. Hydroxyl radical footprinting identified the strongest site for binding of HMG-I(Y), and presumably for the other proteins as well, to be at the center of 4H. Together these in vitro results demonstrate that HMG-I(Y) and H1 are co-dominant over HMG-1 for binding to the central crossover region of 4H and suggest that in vivo both of these proteins may exert a dominant effect over HMG-1 in recognizing and binding to altered DNA structures, such as Holliday junctions, that have conformations similar to 4H.
Hill
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DA
Department of Biochemistry/Biophysics, Washington State University, Pullman, WA 99164-4660, USA.
Reeves
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R
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D025741:HMGA1a Protein; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D006657:Histones / Q000378:metabolism*; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D011485:Protein Binding
D002352:Carrier Proteins; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D006657:Histones; D025741:HMGA1a Protein; D017665:Hydroxyl Radical; D004247:DNA