An unusual type of genetic mutation-trinucleotide repeat expansion-causes Huntington's disease and 14 other neurodegenerative disorders. The first disease-causing TNR expansions were reported only in 1991, so there is still much to learn about their mechanistic roots. In addition to their medical relevance, the genetics of trinucleotide repeats (TNRs) are unique and complex. It is now clear that TNR expansions and contractions occur by multiple genetic mechanisms, including aberrant DNA replication, repair, and possibly gene conversion. The goal of this work is to define more thoroughly how DNA replication and repair contribute to TNR instability in yeast and in primate (human and simian) cells. Mechanistic similarities between yeast and primate cells will help delineate important fundamental properties that govern triplet repeat alterations. For example, the identification of yeast genetic pathways affecting TNRs should help clarify the roles of homologous human pathways. Differences between yeast and primate cells may help resolve certain issues, such as the strong tendency towards expansions in humans which has not been recapitulated in model systems. One unique facet of our proposal is to better understand thresholds. The threshold is a distinctive but enigmatic feature of TNRs where instability changes dramatically over a narrow range of tract lengths. We detected thresholds in both yeast and primate cells, and we propose to dissect them genetically. To help achieve our goal, we developed genetic assays for the direct selection of TNR expansions or contractions. Repeats are inserted into a promoter-reporter construct such that the TNR length determines reporter gene expression. Variations in TNR length are revealed as changes in the reporter phenotype. These selective assays provide several major advantages, including sensitivity, quantitation, and flexibility. We believe the innovative nature of our genetic assays, and our application of those assays to important model systems, will continue to help advance the field of TNR genetics.
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