Mutations at position 277 modify the DNA-binding specificity of human p53 in vitro

p53 regulates the expression of different genes that contain in their promo ter a DNA sequence with two copies of the 16-base motif Pu1Pu2Pu3C4(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 d etermine whether p53 could be mutated at position 277 so that it binds pref erentially to a sequence containing thymine or cytidine. Various mutant pro teins 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 hydroge n bond with the cytidine but not with the thymine. (C) 1999 Academic Press.