Instability of CAG and CTG Trinucleotide Repeats in Saccharomyces cerevisiae

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 3 10 24 to 18 3 10 24 per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3 ,o rPMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast. Tandemly repeated simple sequences are widely distributed throughout the genomes of many eukaryotic organisms. Simple repeats are typically arranged in tracts with 1 to 5 nucleotides (nt) per repeating unit (33). The mutability of these sequences in several human genetic diseases has engendered special interest in the cellular factors governing repeat stability. Mismatch repair provides an important role in stabilization of mononucleotide and dinucleotide repeats. Bacteria, yeasts, and mammalian cells deficient in mismatch repair exhibit increases of up to 700-fold in the rate of dinucleotide repeat instability (1, 10, 18, 31, 32, 34). The mutational spectrum of altered dinucleotide tracts in mismatch repair mutants is dominated by expansions and contractions of one or two repeat units. In wild-type backgrounds, about 10% of the alterations are contractions of five to eight repeats. The observation of dinucleotide repeat instability in Saccharomyces cerevisiae mismatch repair mutants (31, 32) and in cells derived from certain human colon tumors (1, 10, 34) was one of the key clues leading to the discovery that hereditary nonpolyposis colon cancer is due to defects in mismatch repair (4, 22). The genetic stability of trinucleotide repeats differs from that of dinucleotide repeats, as inferred from the mutational basis of several human diseases. Expansions that increase the lengths of naturally occurring trinucleotide tracts are the genetic basis for Huntington’s disease, fragile X syndrome, myotonic dystrophy, and several other diseases (reviewed in references 2, 7,and 23). Among the affected kindreds, expansions are frequent events that lead to the large numbers of trinucleotide repeats associated with the disease state. Although disease-causing mutations were originally believed to arise solely