Factors Controlling Minisatellite Stability in Yeast

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DESCRIPTION (provided by applicant): Maintenance of genome stability is of paramount importance; genome instability has been correlated with numerous disease states in humans, for example. Repetitive DNA is often the source of genomic rearrangements. One class of repetitive DNA, minisatellite sequences, have an approximate repeat unit length ranging from 15 to 100 nucleotides. Mammalian genomes contain a large number of different minisatellite tracts. While these tracts are stable during the mitotic cell cycle, they destabilize during meiosis, altering in both length and sequence composition. Unfortunately the genetic and physical factors controlling the stability of minisatellite tracts are unknown. Minisatellite tracts can have genetic functions; a human minisatellite tract associated with the HRAS1 oncogene acts as a transcription enhancer for HRAS1, and altered minisatellite alleles have been correlated with HRAS1 oncogenesis. We established a model system by introducing the HRAS1 minisatellite into the HIS4 locus in the yeast S. cerevisiae, where it exhibits all of the phenotypes observed in mammalian cells. The tract stimulates transcription and meiotic recombination, and undergoes meiosis-specific alterations in length. Removal of a recombination-initiating endonuclease eliminates tract alterations, while removal of a meiotic DNA loop repair pathway specifically reduces the frequency of tract expansions. These initial studies will be extended by determining the complete complement of genes governing stability of the HRAS1 minisatellite. Two general screens for minisatellite stability maintenance genes have been initiated, in addition to a directed screen of the Yeast Deletion Strain Bank. The screens will identify genes required for minisatellite stability as well as genes affecting DNA loop repair. Finally, we will determine the basis for an observed correlation between HRAS1 minisatellite allele state and breast cancer oncogenesis, through genetic analysis of native human HRAS1 minisatellite alleles. Many of 1he proposed experiments cannot be done in a mammalian system; the HIS4-HRAS1 minisatellite system is the only means to gain these important data. These studies will provide insights into genome maintenance, recombination, DNA repair, transcription initiation, and aspects of cancer predisposition.