Verrijzer C P, Kal A J, van der Vliet P C
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Genes Dev. 1990 Nov;4(11):1964-74. doi: 10.1101/gad.4.11.1964.
The ubiquitous octamer-binding protein oct-1 contains a POU domain required for DNA binding, which can be subdivided into a POU-specific domain and a POU homeo domain. We have overproduced the POU domain and the POU homeo domain in a vaccinia expression system, purified both polypeptides to near homogeneity, and compared their DNA-binding properties. In contrast to the POU domain, the homeo domain protects only part of the octamer sequence in the Ad2 origin against breakdown by DNase I or hydroxyl radicals. Analysis of purine contacts by DMS and DEPC interference assays shows that the Ad2 octamer can be divided into two regions: one that is recognized both by the POU domain and the homeo domain in an identical fashion, and one that is only recognized by the POU domain. This suggests that the POU-specific domain is responsible for the additional contacts located at one side of the octamer. In agreement with this, mutating the first 3 nucleotides (ATG) of the octamer affected binding by the POU domain but not by the homeo domain. The apparent binding affinities to different octamer sites were compared. The homeo domain binds 600-fold less efficiently to the canonical octamer sequence (ATGCAAAT) than the POU domain. The difference is only sevenfold for the Ad2 octamer, whereas both Kd values are almost identical for the HSV ICP4 TAATGARAT motif. Both the POU and homeo domains recognize target sequences for mammalian homeo box proteins. We conclude that the octamer can act as a bipartite recognition sequence for oct-1 and that the POU-specific domain contributes to the binding affinity, as well as to the specificity, by providing additional contacts.
普遍存在的八聚体结合蛋白Oct-1含有一个DNA结合所需的POU结构域，该结构域可细分为一个POU特异性结构域和一个POU同源结构域。我们已在痘苗病毒表达系统中过量表达了POU结构域和POU同源结构域，将这两种多肽纯化至近乎均一，并比较了它们的DNA结合特性。与POU结构域不同，同源结构域仅保护腺病毒2型（Ad2）起始位点中八聚体序列的一部分免受核酸酶I或羟自由基的破坏。通过二甲基亚砜（DMS）和焦碳酸二乙酯（DEPC）干扰试验对嘌呤接触进行分析表明，Ad2八聚体可分为两个区域：一个区域以相同方式被POU结构域和同源结构域识别，另一个区域仅被POU结构域识别。这表明POU特异性结构域负责八聚体一侧的额外接触。与此一致的是，八聚体的前3个核苷酸（ATG）发生突变会影响POU结构域的结合，但不影响同源结构域的结合。比较了对不同八聚体位点的表观结合亲和力。同源结构域与典型八聚体序列（ATGCAAAT）的结合效率比POU结构域低600倍。对于Ad2八聚体，差异仅为7倍，而对于单纯疱疹病毒1型（HSV）ICP4的TAATGARAT基序，两个解离常数（Kd）值几乎相同。POU结构域和同源结构域均识别哺乳动物同源框蛋白的靶序列。我们得出结论，八聚体可作为Oct-1的双部分识别序列，并且POU特异性结构域通过提供额外接触有助于结合亲和力以及特异性。
