Interaction of the phd-finger homeodomain protein hat3.1 from arabidopsis thaliana with dna. specific dna binding by a homeodomain with histidine at position 51

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Biochemistry 2007, 46, 7416-7425

Interaction of the PHD-Finger Homeodomain Protein HAT3.1 from Arabidopsis thaliana with DNA. Specific DNA Binding by a Homeodomain with Histidine at Position 51†
Ivana L. Viola and Daniel H. Gonzalez*
Catedra de Biologıa Celular y Molecular, Facultad de Bioquımica y Ciencias Biologicas, UniVersidad Nacional del Litoral, ´ ´ ´ ´ CC 242 Paraje El Pozo,3000 Santa Fe, Argentina ReceiVed December 28, 2006; ReVised Manuscript ReceiVed April 3, 2007

ABSTRACT: HAT3.1 is a member of the PHD-finger homeodomain protein family. The HAT3.1 homeodomain is highly divergent in sequence even at positions that are almost invariable among homeodomains. In this work, we have applied the random oligonucleotide selection technique to investigate if the HAT3.1homeodomain is able to recognize specific DNA sequences. Analysis of the selected molecules followed by hydroxyl radical footprinting experiments and yeast one-hybrid assays indicated that HAT3.1 shows a preference for the sequence T(A/G)(A/C)ACCA, different from those bound by other homeodomains. Binding was dependent on homeodomain residues located at positions 47, 50, 51, and 54, the samepositions that usually participate in DNA binding in most homeodomains. The study of the interaction of mutants at these positions with DNA carrying nucleotide changes at specific sites suggested that H51 and K50 most likely interact with nucleotides 2 to 4 and 5 to 6, respectively, while W54 would establish contacts with position 4. The presence of H51 and W54 represents an innovation among homeodomainstructures. The fact that the HAT3.1 homeodomain is able to interact with specific DNA sequences is evidence of the inherent plasticity of the homeodomain as a DNA binding unit.

The homeodomain (HD1) is a 60 amino acid motif found in a vast group of eukaryotic transcription factors. Its name comes from the study of homeotic mutations in Drosophila, which produce important alterations as aresult of the development of tissues or organs in wrong places. HDcontaining proteins have now been found in almost every eukaryotic organism that was searched for them, and a common theme seems to be that they participate in the regulation of developmental processes (1-3). From X-ray diffraction and NMR studies, a general model for the structure of the HD has emerged (4-6). Briefly, it is conformedby three R-helices connected by a loop (helices I and II) and a turn (helices II and III). Helices I and II are antiparallel, and helix III is perpendicular to the other two. The structure is maintained by hydrophobic contacts between conserved residues from the different helices. The first nine residues are disordered and constitute the N-terminal arm. The fact that helices II and III form astructure that resembles the helix-turn-helix motif found in prokaryotic transcription factors suggested for the first time that the HD may function in DNA binding (7). Protein-DNA interaction studies have shown that this is indeed the case. Specific contacts with DNA are established by residues located within helix III and in the N-terminal arm (8-11). Nonspecific contacts are also
† Supported bygrants from CONICET, ANPCyT, and Universidad Nacional del Litoral. D.H.G. is a member of CONICET; I.L.V. is a fellow of the same Institution. * To whom correspondence should be addressed. Phone/fax: 54342-4575219. E-mail: dhgonza@fbcb.unl.edu.ar. 1 Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; GST, glutathione S-transferase; HD, homeodomain.

established by residues in helices IIand III and in the loop. A majority of HDs bind the sequence TAATNN, where the first two residues are contacted by the N-terminal arm, while the third and fourth are recognized by the almost invariant N51 and highly conserved isoleucine or valine at position 47, respectively (11). The next two positions are more variable and are contacted by residues 50 and 54, which are considerably less...
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