Expansions of trinucleotide (triplet) repeats at specific sites in the human genome cause a number of neurological diseases, including myotonic dystrophy, Huntington disease, fragile X syndrome, and several others. At the myotonic dystrophy locus, normal individuals have up to 30-40 CTG/CAG repeats, whereas affected individuals may have up to several thousand repeats. CTG/CAG repeats have a propensity to form a variety of stable secondary structures in vitro, and it is thought that these unusual structures interfere with aspects of DNA metabolism in cells, leading to repeat expansion and disease. Studies in E. coli and S. cerevisiae have shown that triplet repeat stability is sensitive to processes that expose single strands of DNA, including transcription, replication, repair, and recombination. This application seeks to develop novel selective systems for investigation CTG/CAG triplet repeat stability in vertebrate cells. Instability during recombination and the effects of triplet repeats on the recombination processes, which have already been demonstrated at the APRT locus in CHO cells, will be defined using a variety of tandem duplication substrates, I-SceI-mediated double-strand breaks, and ERCC1-deficient cells. Instability of repeats due to all causes will be investigated using a novel, direct-selection assay based on the length-dependent effects on gene expression by intronic CAG repeats, which have already been demonstrated. Long repeats placed in the intron of the HPRT minigene, which render it HPRT , can be used to select for repeat contractions (HPRT- to HPRT+). Similarly, short CAG repeats that are compatible with gene expression can be used to select for repeat contractions (HPRT- to HPRT+). Similarly, show CAG repeats that are compatible with gene expression can be used to select for expansions (HPRT- to HPRT+). The boundaries for these length- dependent effects will be defined, the mechanism of interference will be determined, and appropriate CAG-containing, HPRT-minigene substrates will be deposited in the chromosomes of vertebrate cells. These substrates will allow testing of the effects of genes involved in replication, repair, and in recombination and cell treatments that stress these processes. In summary, we propose and integrated and comprehensive set of experiments to define the molecular basis of the CTG/CAG repeat instability that underlies myotonic dystrophy and other neurological diseases. These studies will also provide a set of experimental reagents that will e useful in the design and evaluation of potential therapeutic strategies directed at preventing expansion or promoting contraction of CTG/CAG repeats.
