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 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.
Chène
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Oncology Department, Novartis, Basel, CH-4002, Switzerland.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003562:Cytidine / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016158:Genes, p53; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013941:Thymine / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D003562:Cytidine; D004247:DNA; D013941:Thymine