UV-induced mutagenesis of human p53: analysis using a double-selection method in yeast.

Comparison of the mutation patterns of p53 in human tumors with those of selectable genes in model systems is a powerful approach to identify potential etiological factors for specific tumor types. Recently, we validated use of a yeast assay to permit direct determination of the mutation spectrum induced in human p53 by carcinogens that would reduce uncertainties inherent in comparing spectra induced in different target genes. Here, we describe modifications in the assay designed to facilitate screening for mutants and to permit intracellular exposure of the gene instead of in vitro treatment. This was accomplished by introducing growth-based selection for transactivation-deficient p53 mutants into yeast already possessing red/white colony color selection. This improved model system was able to detect cells harboring p53 mutations among cells with wild-type p53 at a frequency of 10(-4) or less. Additionally, UV light was used to verify that the majority of mutagenized cells with the appropriate phenotype on selective medium contained mutations in p53, not elsewhere in the genome. Sequence analysis of UV-induced mutations revealed that the nature of the mutations was similar to those obtained in previous studies of this mutagen. This system will prove useful in the determination of the ability of environmental agents to mutate the human p53 gene, and thus may contribute to hazard identification.

UV-induced mutagenesis of human p53 in a vector replicated in Saccharomyces cerevisiae.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration identified to date in human cancers. Similarities of p53 mutations found in human cancers with those induced in experimental systems have been interpreted as evidence supporting a causative role for environmental carcinogens in certain tumor types. We have developed and validated a method for generation of mutation spectra and measurement of mutation frequency directly on human p53 cDNA in a vector following treatment with mutagens and replication in yeast. Mutants that had lost the DNA binding/transcription activation function of p53 were detected by yeast colony color, isolated, and sequenced. UV light was used to characterize and validate the system, and a dose-dependent increase in mutation frequency was seen following exposure of the plasmid to increasing doses of UV, resulting in an 18-fold increase over the spontaneous frequency (3.2 x 10(-4)) at the highest level tested (300 J/m2). Sequence analysis of p53 in the mutants revealed that the types of mutations induced were similar to those obtained in previous studies of UV mutagenesis in other model systems, and the types and positions of mutations were also similar to those found in human skin tumors. This experimental system will be useful in further evaluation of the importance of environmental agents as risk factors for cancer.