Analysis of mutant p53 for MAR-DNA binding: determining the dominant-oncogenic function of mutant p53

摘要

At least some mutant p53 proteins not simply have lost the wild-type p53 specific tumor suppressor function, but exhibit oncogenic functions on their own. Recently we showed that binding of mutant p53 to MAR/SAR elements is an activity specific for mutant p53 and clearly distinguishable from the previously reported DNA-binding activities of p53. Since MAR/SAR elements are considered to be important regulatory elements for a variety of nuclear processes, the interaction of mutant p53 with MAR/SAR elements might form the molecular basis for oncogenic potential of mutant p53. By employing different binding assays (the target-bound DNA binding assay, the South-western blotting technique and an adapted liquid phase binding assay), we studied MAR/SAR binding of various p53 proteins to different MAR/SAR elements. Murine mutant p53 bound different MAR/SAR elements with an approximately 1,000-fold higher affinity than murine wild-type p53. Analysis of MAR/SAR binding of human wild-type and mutant p53 proteins revealed also high affinity MAR/SAR binding of several human p53 mutant proteins (175 Arg His, 273 Arg Pro), but not of human wild-type p53, confirming that MAR binding is a general property of mutant p53. By antibody interference analysis using a panel of different p53- specific monoclonal antibodies and by deletion mutant analysis the MAR/SAR binding domain on mutant p53 was mapped, revealing a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids.