Chène P
Oncology Department, Novartis, Basel, CH-4002, Switzerland.
Biochem Biophys Res Commun. 1999 Sep 16;263(1):1-5. doi: 10.1006/bbrc.1999.1294.
p53 regulates the expression of different genes that contain in their promoter a DNA sequence with two copies of the 10-base motif Pu(1)Pu(2)Pu(3)C(4)(A/T)(5)(T/A)(6)G(7)Py(8)Py(9)Py(10). This sequence is degenerated, and thymine or cytidine is found equally at position 3 or 8. These two bases make contact with cysteine-277 of the human p53. An in vitro study was carried out to determine whether p53 could be mutated at position 277 so that it binds preferentially to a sequence containing thymine or cytidine. Various mutant proteins were created and their DNA-binding specificity was determined by gel shift assay. Two of them show an altered specificity. The Cys277Ser protein binds preferentially to cytidine-containing sequences while the Cys277Ala mutant has a preference for thymine-containing sequences. This specificity is presumably achieved because an alanine residue at position 277 interacts with the thymine via hydrophobic interactions and a serine makes a hydrogen bond with the cytidine but not with the thymine.
p53调控不同基因的表达，这些基因的启动子中含有一段DNA序列，该序列有两个拷贝的10碱基基序Pu(1)Pu(2)Pu(3)C(4)(A/T)(5)(T/A)(6)G(7)Py(8)Py(9)Py(10)。此序列是简并的，在第3位或第8位胸腺嘧啶或胞嘧啶出现的概率相同。这两个碱基与人类p53的半胱氨酸-277接触。开展了一项体外研究，以确定p53在第277位是否会发生突变，从而使其优先结合含有胸腺嘧啶或胞嘧啶的序列。构建了各种突变蛋白，并通过凝胶迁移试验确定其DNA结合特异性。其中两种显示出特异性改变。Cys277Ser蛋白优先结合含胞嘧啶的序列，而Cys277Ala突变体则偏好含胸腺嘧啶的序列。推测这种特异性的实现是因为第277位的丙氨酸残基通过疏水相互作用与胸腺嘧啶相互作用，而丝氨酸与胞嘧啶形成氢键，但不与胸腺嘧啶形成氢键。
