To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3 end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 ؋ 10 ؊2 changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5 to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5 end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.Expansions of repetitions of the trinucleotide CAG are the cause of a number of human inherited, dominant neurological and neuromuscular diseases, including Huntington's disease (14), two forms of spinocerebellar ataxia (type 1 and MachadoJoseph disease) (17,20), and myotonic dystrophy (3,7,19). Although CAG trinucleotide repetitions are present in normal alleles of the genes giving rise to these disorders, mutant alleles have tracts which are longer than those within the normal range. The long tracts within disease alleles are unstable in that children often inherit a repeat length different from that found in their affected parent. The instability most likely reflects replicative errors which occur either during the meiotic division of gametogenesis or during the mitotic divisions preceding it.The underlying cause of the instability is thought to reflect the ability of CAG repeats to form palindrome-like structures (8,22). Such structures may present problems to the replication fork as it passes through them. One study using small CAG repeats embedded in palindromes carried on phage lambda showed that they were inhibitory to plaque formation (6). Studies with Escherichia coli have also shown that CAG repeats undergo both contractions and expansions when propagated in a bacterial plasmid (16).We decided to examine the stability of CAG repeats in Saccharomyces cerevisiae because the chromatin structure and chromosomal replication of this simple eukaryote have many similarities to the chromosomal mechanics of more complex euka...

To explore the mechanisms by which CAG trinucleotide repeat tracts undergo length changes in yeast cells, we examined the polarity of alterations with respect to an interrupting CAT trinucleotide near the center of the tract. In wild-type cells, in which most tract changes are large contractions, the changes that retain the interruption are biased toward the 3 end of the repeat tract (in reference to the direction of lagging-strand synthesis). In rth1/rad27 mutant cells that are defective in Okazaki fragment maturation, the tract expansions are biased to the 5 end of the repeat tract, while the tract contractions that do not remove the interruption occur randomly on either side of the interruption. In msh2 mutant cells that are defective in the mismatch repair machinery, neither the small changes of one or two repeat units nor the larger contractions attributable to this mutation are biased to either side of the interruption. The results of this study are discussed in terms of the molecular paths leading to expansions and contractions of repeat tracts.

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