MUTATION OF CONSERVED DOMAIN-II ALTERS THE SEQUENCE SPECIFICITY OF DNA-BINDING BY THE P53 PROTEIN

We have mutagenized human p53 expressed in yeast and selected two muta nts, 121F and 123A, which activate transcription from one, rather than the normal two, copies of the consensus p53 DNA binding sequence. Bot h mutants have a 6-fold increase in affinity for a single copy of the sequence GGG CATG CCC. The 121F mutant has a decrease, and the 123A mu tant an increase, in the affinity for the sequence GAA CATG TTC. This genetic and biochemical evidence supports the crystallographic finding that amino acid 120 contacts guanine in the major groove at the secon d position in the consensus. The major p53 binding site in the p21(WAF 1/CIP1) promoter resembles the GAA CATG TTC form of the consensus. Com pared with wild type p53, the 121F mutant has a 7-fold lower affinity for the p21(WAF1/CIP1) site in vitro, and the 121F mutant is defective in p21 induction in vivo. Mutants with subtly altered sequence specif icity may facilitate dissection of downstream pathways activated by p5 3.