of Work: Yeast based assays provide for the functional characterization of human genes that may play a role in chromosome metabolism and genome stability. This led to the isolation of the human cyclin A1 gene due to a no-growth phenotype in a replication mutant (pol3-t) and subsequent characterization of human cyclin A1. We showed that it interferes with yeast cyclin-dependent checkpoint controls. Complementation of a yeast rad27 mutant and synthetic lethality was exploited to develop functional assays for the human flap endonuclease hFEN which is involved in DNA replication and repair. The identification of a biochemically inactive form of hFEN1 led to the development of a screening assay for toxic Fen1 mutants (also see Project 65073). The transactivation capabilities of human p53 genes are being investigated with our recently developed yeast assay and this is being used to evaluate p53 status in tumor material (in collaboration with the Taylor lab) by direct recombinational cloning and expression in yeast. The p53 tumor suppressor gene acts as a potent transcription factor and exhibits sequence specificity in DNA binding and different affinities toward promoter elements. We exploited variable expression and different p53 responsive elements in the yeast-based transactivation assay to isolate and characterize novel p53 alleles. We identified alleles that were toxic at moderate expression levels and alleles with greater than wild type levels of transactivation (""""""""supertrans""""""""). These mutants are useful for understanding structure-function relations for the many different response elements as well helping assess how p53 mutations may give rise to cancer. These novel p53 mutants will be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and DNA, and the development of cancer therapies.
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