Destabilization of CAG trinucleotide repeat tracts by mismatch repair mutations in yeast
Destabilization of CAG trinucleotide repeat tracts by mismatch repair mutations in yeastJill Kuglin Schweitzer and Dennis M. Livingston*
Department of Biochemistry, 4-225 Millard Hall, 435 Delaware Street SE, University of Minnesota, Minneapolis, MN 55455-0347, USA
Received September 11, 1996;Revised and Accepted December 6, 1996
To examine the genetic factors that affect the stability of disease-associated trinucleotide repeats, we have assessed the stability of CAG repeats in yeast strains with mutations in the mismatch repair system. We have found that both pms1 and msh2 mutations destabilize repeat tracts. Destabilization is evidenced both by the increased frequency of repeat length changes and in the pattern of changes that are observed. In wild-type cells repeats are relatively stable when CAG serves as the lagging strand template but relatively unstable when CTG serves as the lagging strand template. Large contractions in repeat length are the most common change. In pms1 and msh2 mutants the relatively stable tracts incur more tract length changes. In addition, many small deletions and some small additions, most often of one repeat unit, are frequent in repeats of the stable orientation. These small changes also are seen as a new class of events that occur in repeats in the unstable orientation. The results show that in yeast the mismatch repair system prevents small changes from occurring but cannot prevent larger changes from occurring.
Repetitions of the trinucleotide CAG are the cause of a number of inherited neurological and neuromuscular diseases. A prominent feature of these diseases is that the number of repeat units often changes when passed from parent to child. Although changes occur when either the mother or father is the origin of the disease allele, there is often a sex bias in the parent that gives rise to a predominance of expansions. In congenital cases of myotonic dystrophy caused by very large repeat tracts the disease allele is most often provided by the mother (1 -4 ). In Huntington's disease and Spinocerebellar Ataxia Type 1 increases most often occur when the disease allele is passed on by the father (5 ,6 ). In Machado-Joseph disease changes in repeat number often occur, but a bias towards larger or smaller sizes is not readily apparent (7 -10 ). What factors control these differences among disease genes is unknown.
We have been studying the instability of CAG repeats embedded in a yeast chromosome (11 ). CAG repeats are unstable in yeast giving rise to frequent large contractions and rarer increases in repeat number. We have already shown that the orientation of the repeat with respect to a replication origin has a 10-fold effect on its stability. When CTG, the complement of CAG, serves as the lagging strand template the repeat is more unstable than when CAG serves as the lagging strand template.
We have also begun to examine the repair, replication and recombination systems needed to maintain repeat stability. One repair system that is known to destabilize repeats of the dinucleotide GT in both humans and yeast is the mismatch repair system (12 ). In this study we show that CAG repeats in yeast are destabilized by mutations in two mismatch repair genes, pms1 and msh2.
The yeast strains we use to study the stability of trinucleotide repeat tracts contain insertions of CAG repeat tracts into chromosome VII (Fig. 1 ) (11 ). This was achieved by appending repeat tracts to the yeast ADE2 gene and using this gene to disrupt the ARO2 gene on chromosome VII. Both orientations of the repeat, CAG and CTG, as well as both orientations of ADE2 have been made. We previously showed that repeat tracts in the two strains with CTG in the ADE2 coding strand, tracts D and H, are relatively unstable compared to repeat tracts C and Q with CAG in the ADE2 coding strand. The difference in stability arises because the ADE2 clone contains a replication origin, and the greatest instability occurs when CTG serves as the lagging strand template. Most changes in yeast are deletions that are >15 repeat units.
Instability can be assessed by determining the percentage of cells within a colony that contain a repeat tract length different from the tract length found in the progenitor cell. To make this assessment, colonies are dispersed into individual cells, the sibling cells are permitted to grow into colonies, and DNA samples from the sibling colonies are analyzed to see whether or not they contain the parental size repeat. The frequency of colonies lacking the parental size repeat is a metric of the instability. Additional information can be derived by measuring the difference in size between the parental repeat and the new, smaller (or larger) repeat tract.
When repeat tracts are oriented such that CAG serves as the lagging strand template, the repeat tracts are relatively stable in wild-type cells, yielding only a few sibling colonies that do not contain the parental size repeat (Table 1 ). In pms1 and msh2 mutant cells these tracts are more unstable (Table 1 ). For example, tract C (85 repeat units) changed in size in two of 59 sibling colonies in a wild-type background, whereas in the pms1 mutant the number was 12 out of 59 (combined 75 and 85 repeat units) (Table 1 , Fig. 2 ) and in the msh2 mutant the number that changed was 17 out of 59 (combined 75 and 85 repeat units) (Table 1 , Fig. 3 ). [These frequency measurements can be converted to rates using the method of Lea and Coulson (13 ) by assuming that the frequencies are median values (11 ). The rate values for wild type, pms1 and msh2 cells are 4 * 10-3, 2.2 *10-2 and 3.0 * 10-2 changes/cell/generation, respectively.] A further indication of the increased instability in mismatch mutants comes from examination of strains with short tract lengths. Whereas 28 repeat units of tract C are stable in wild-type cells (0 out of 60), this small tract undergoes length changes in mutant cells (5 of 60 for pms1; 1 of 60 for msh2) (Table 1 ).
More striking is the spectrum of length changes in mutant cells (Table 1 ). A large percentage of events that occur in mutant cells are deletions or additions of one repeat unit, a class of events that are not seen in wild-type cells. Among the events from pms1 cells containing tract C (tracts of 75 and 85 repeats), eight out of 12 events were changes of <= 3 repeat units, and of the eight small changes six were deletions (Table 1 , Fig. 2 ). Among the events from msh2 cells containing tract C (tracts of 75 and 85 repeats), 15 out of 17 events were changes of <= 2 repeat units, and of those 14 were deletions (Table 1 , Fig. 3 ). These results show that both pms1 and msh2 mutations destabilize tracts that are mostly stable in wild-type cells.
When long repeat tracts are oriented such that CTG serves as the lagging strand template, the repeat tracts are relatively unstable in wild-type cells, yielding between 15 and 25% of sibling colonies that do not contain the parental size repeat (Table 1 ). Among such colonies most tract length changes are relatively large decreases. For example, the eight events undergone by tract D (69 repeat units) ranged from decreases of 23 to 54 repeat units (Table 1 ).
In mutant cells these tracts continue to undergo large deletions (Table 1 , Fig. 4 ). In addition they exhibit the class of events seen for the relatively stable tracts in which changes of one or a few repeat units occur. For example, the pms1 strain containing tract D (69 repeats) underwent eight changes, half of which were deletions of 1 unit. The msh2 strain containing tract D (69 repeats) underwent 14 changes of which four were small deletions or additions of 1 or 2 units.
The mismatch repair mutations, pms1 and msh2, destabilize CAG repeat tracts in yeast. The destabilization is best shown by the increased instability of tracts that are relatively stable in wild-type cells. In addition, a new class of events occurs in mutant cells in which tracts change by one or a few repeat units. Most of these changes are decreases.
That the mismatch repair system should have an effect on CAG repeat tract stability is not surprising considering its involvement in stabilizing dinucleotide tracts (14 ,15 ). The behavior of trinucleotide repeats in the mismatch mutants offers additional insights into this system in yeast. In particular, the addition of a new class of events with very small changes indicates that the mismatch repair system is very proficient in preventing such small changes. The observation that most changes in wild-type cells are large deletions suggests that once a mismatch increases beyond a size of one or a few repeats, the mismatch repair system cannot correct the change (Fig. 5 ). This corroborates recent studies on deletions of direct repeats (16 ) as well as earlier studies of mismatch correction of artificial substrates in vivo and in vitro (17 ,18 ).
Yeast strain SSL204A (MATa) with repeat tracts C, D and H have been previously described (11 ). Tract Q is an independent isolate of tract orientation G reported previously. To create isogenic mismatch repair mutants containing repeat tracts, the wild-type strains SSL204A (MATa) and SSL204 (MAT[alpha]), that differ only in mating type, were disrupted with mutant copies of the mismatch repair genes PMS1 and MSH2 (37 -39 ). Plasmids bearing disruptions of PMS1 and MSH2 were obtained from Richard Kolodner (Dana-Farber Cancer Institute). Southern blotting was used to confirm the disruption of the mismatch repair genes in all cases. Two schemes were used to place repeat tracts in the mismatch repair mutants. First, some mutants were transformed directly with the aro2::ADE2(CAG)n disruptions. Second, some were made by mating the pms1 and msh2 mutant derivatives of SSL204 (MAT[alpha]) to the SSL204A (MATa) strain containing the appropriate repeat tract. Sporulation of such diploids yield isogenic, haploid segregants of the appropriate genotype.
The procedures for colony dispersal into single cells and isolation of DNA from sibling colonies have been previously described (11 ). The PCR conditions are a slight variation on the previously reported method. In this study we used 0.375 U Taq polymerase (AmpliTaq, Perkin Elmer) with a buffer that contained 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2, 2% v/v formamide, 250 [mu]M each dNTPs and 1 [mu]M each of primers DMLAde2b and DMLAde2c.
In this study many small changes of 1 or 2 repeat units were detected in tracts as long as 92 repeat units. When such small changes were detected, the products were run on a second gel in the proximity of appropriate control samples to verify their sizes.
This work was supported by grant P01NS33718 from the National Institutes of Health. We thank Debra Maurer and Brennon O'Callaghan for technical help and Richard Kolodner (Dana-Farber Cancer Institute) for supplying disruptions of PMS1 and MSH2. We thank Harry Orr for his encouragement and for his comments on the manuscript.
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*To whom correspondence should be addressed
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