): The ankyrin (ANK) repeat is a thirty-three amino acid motif present in many proteins important for cellular growth and differentiation. The thirty-three amino acid ANK repeat motif was first described in proteins involved in cell cycle and fate-determination proteins and since then have been found in a number of proteins from nearly all phyla. There is considerable variation in the consensus sequence for the ANK motifs. The ANK repeats are thought to be involved in protein:protein interaction, however, the function of the repeats is not known. Increasing evidence suggests a direct role for ANK repeats in cellular transformation. The ANK repeat regions in BCL-3, int-3, TAN-1, and p16 have all been implicated in tumor development. Both BCL-3 and TAN-1 are involved in gene rearrangements specific to B-cell chronic lymphocytic leukemias and T-cell acute lymphoblastic leukemias, respectively. Mutations in the human p16 protein have been identified in many tumor cell lines, in some primary tumors, and recently in germline DNA of some families predisposed to melanoma. The ANK repeat regions of the mouse proto-oncogene, int-3, are necessary and in themselves sufficient to cause mammary and salivary adenocarcinomas in mice. The persistent involvement of the ANK repeats in tumorigenicity necessitates a thorough analysis of these structures. Genetic and biochemical analysis of the ANK repeat regions of a functionally well-characterized protein could contribute significantly to the understanding of aberrant molecular and cellular processes.
The aim of this proposal is to define the function of the ANK repeats and to determine the residues within the motif that convey functional significance. The investigators propose to genetically analyze the function of the ANK repeat domains of Swi6, a cell cycle regulatory protein in the budding yeast, Saccharomyces cerevisiae. Using oligonucleotide site-directed mutagenesis, they will target the SWI6 ANK domains for mutagenesis and screen for temperature-sensitive Swi6 ANK mutants by detecting activation of HO transcription. Using genetic and biochemical screens, they can identify the residues of the ANK repeat regions that are necessary for Swi6 protein stability and function. The investigators anticipate that this analysis will be important for higher eukaryotic cells as the ANK repeats are widely conserved motifs. In addition, Swi6 is an important regulator of growth control in yeast, and information that relates ANK function to this action of Swi6 will contribute on a basic level to understanding the controls of eukaryotic cell growth.
