Viola Ivana L, Gonzalez Daniel H
Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina.
Biochemistry. 2007 Jun 26;46(25):7416-25. doi: 10.1021/bi602657e. Epub 2007 May 26.
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 HAT3.1 homeodomain 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 same positions 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 homeodomain structures. 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.
HAT3.1是PHD指同源结构域蛋白家族的成员。HAT3.1同源结构域在序列上高度不同，即使在同源结构域中几乎不变的位置也是如此。在这项工作中，我们应用随机寡核苷酸选择技术来研究HAT3.1同源结构域是否能够识别特定的DNA序列。对所选分子进行分析，随后进行羟自由基足迹实验和酵母单杂交分析，结果表明HAT3.1对序列T(A/G)(A/C)ACCA有偏好，这与其他同源结构域结合的序列不同。结合依赖于位于第47、50、51和54位的同源结构域残基，这些位置通常是大多数同源结构域中参与DNA结合的相同位置。对这些位置的突变体与在特定位点携带核苷酸变化的DNA之间相互作用的研究表明，H51和K50最有可能分别与第2至4位和第5至6位的核苷酸相互作用，而W54将与第4位建立接触。H51和W54的存在代表了同源结构域结构中的一项创新。HAT3.1同源结构域能够与特定DNA序列相互作用这一事实证明了同源结构域作为DNA结合单元具有内在的可塑性。
