The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.     

The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin

The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.     

High affinity insertion/deletion lesion binding by p53. Evidence for a role of the p53 central domain

In addition to binding DNA in a sequence-specific manner, p53 can interact with nucleic acids in a sequence-independent manner. p53 can bind short single-stranded DNA and double-stranded DNA containing nucleotide loops; these diverse associations may be critical for p53 signal transduction. In this study, we analyzed p53 binding to DNA fragments containing insertion/deletion mismatches (IDLs). p53 required an intact central domain and dimerization domain for high affinity complex formation with IDLs. In fact, the C terminus of p53 (amino acids 293-393) was functionally replaceable with a foreign dimerization domain in IDL binding assays. From saturation binding studies we determined that the KD of p53 binding to IDLs was 45 pM as compared with a KD of 31 pM for p53 binding to DNA fragments containing a consensus binding site. Consistent with these dissociation constants, p53-IDL complexes were dissociated with relatively low concentrations of competitor consensus site-containing DNA. Although p53 has a higher affinity for DNA with a consensus site as compared with IDLs, the relative number and availability of each form of DNA in a cell immediately after DNA damage may promote p53 interaction with DNA lesions. Understanding how the sequence-specific and nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how signaling cascades are initiated after DNA damage.