DISCRIMINATION OF DNA-BINDING SITES BY MUTANT P53 PROTEINS

Critical determinants of DNA recognition by p53 have been identified b y a molecular genetic approach, The wild-type human p53 fragment conta ining amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA bi nding assays, and full-length human p53 was used for transactivation a ssays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically alt ered forms of a known consensus DNA site. This refinement indicates th at p53 binds with high affinity to two repeats of PuGPuCA. TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A /T).(T/A) GPyPyPy. These results were further confirmed by transactiva tion assays of yeast by using full-length human p53 and systematically altered DNA sites, Dimers of the pentamer AGGCA oriented either head- to-head or tail-to-tail bound efficiently, but transactivation was fac ilitated only through head-to-head dimers, To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutio ns were made at several positions within the DNA binding domain of p53 , and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gin or Arg, and Arg-283 to Gin bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentame r (PuGPuCA), respectively, Thus, we implicate these residues in amino acid-base pair contacts, Interestingly, mutant Cys-277 to Gin bound a consensus site as two and four monomers, as opposed to the wild-type p 53 fragment, which invariably binds this site as four monomers.