1. Mismatch Repair Blocks Expansions of Interrupted Trinucleotide Repeats in Yeast 
Michael L Rolfsmeier, Michael J Dixon, Robert S Lahue 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)

2. Figure 1 Stabilization of Interrupted Alleles by Mismatch Repair
Assay for TNR expansions (Miret et al. 1998) and alleles tested.
(A) The region controlling expression of the reporter gene URA3 is shown, including the TATA box, the TNR region, an out-of-frame initiation codon, the preferred transcription initiation site (I), and the start of the URA3 gene. The upper diagram demonstrates the starting construct. The brackets represent a window of potential transcription initiation sites located 55–125 base pairs from the TATA box. With a 25 TNR tract, the transcription window includes the preferred site I, thus leading to expression of URA3 and making the yeast 5FOA sensitive. The lower diagram demonstrates what happens when the TNR tract expands to ≥30 repeats. The bracketed window does not include site I. Transcription initiating 5′ of the preferred initiation site will include the out-of-frame ATG, resulting in translational incompetence and, therefore, resistance to 5FOA.
(B) The TNR sequences used for this study are shown. Nomenclature is that of the lagging daughter strand. The ATGATG and ATG test sequences are similar to interrupted alleles of SCA1, many of which contain 1–3 CAT interruptions of CAG repeats, resulting in total tract lengths equal to 23–36 repeats (Chung et al. 1993). The complementary strand in SCA1 therefore harbors ATG interruptions in a CTG tract. If ATG interruptions stabilize CTG tracts in yeast as in humans, the rate of expansion should be reduced relative to an uninterrupted control. The importance of the CTCCTC-interrupted allele is explained in the text.
(C) Expansion rates are expressed as events per cell generation × 10−5. All values shown are the average of two to four independent measurements. Rates are typically accurate to within 2-fold, based on measurements of the standard deviations. WT refers to the wild-type parental strain. Fold stabilization refers to the ratio of the expansion rate of the perfect repeat allele divided by the expansion rate for the test situation. Rates for the perfect repeat in wild-type and msh2 strains were taken from Miret et al Rates for ATGATG and central ATGATG in wild type were taken from Rolfsmeier and Lahue 2000.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2 A Coexcision Model for Stabilization
(A) A model for stabilization of interrupted repeats. Open rectangles represent nonrepetitive flanking sequence, the lines denote the TNR, the filled circles symbolize the interruptions, arrowheads mark the 3′ end of the Okazaki fragment, and carats represent mismatches. The model is described in detail in the text.
(B) Possible mispaired hairpin structures. Watson-Crick pairs are denoted by a hyphen. The T nucleotides in the stem are intrahelical and form single H-bond wobble pairs, based on in vitro analysis of CTG repeats (Gacy et al. 1995; Mitas 1997). Note that the ATGATG interruption generates two A-G mispairs separated by two base pairs. Another possible structure for this sequence is a four-base bubble in which the presence of the mispairs destabilizes the intervening base pairs. The ATG interruption yields a single A-G mispair, whereas the CTCCTC yields two C-C mismatches.
(C) 3′ additions and duplications were determined by molecular analysis of the expanded TNR in 5FOAR colonies. PCR products, either undigested or cleaved with SfaNI (for ATG-containing interruptions) or with BseRI (for CTC-containing interruptions), were analyzed on sequencing gels as described (Rolfsmeier and Lahue 2000). Data for ATGATG in wild type were taken from Rolfsmeier and Lahue 2000.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 Limited Stabilization of Contractions
(A) The assay for contractions utilizes a similar strategy as for expansions. The starting TNR tract contains 25 CTG repeats plus 24 base pairs of randomized sequence (“25 + 8”). The total length is therefore equivalent to 33 repeats. This size tract inactivates the URA3 reporter gene, leading to a Ura− phenotype (requires uracil for growth). Contractions that reduce the tract length to final sizes of 0–20 CTG repeats allow utilization of the preferred initiation site I and thereby confer a Ura+ phenotype.
(B) The alleles used for analysis of contractions were the perfect tract and two interrupted alleles in which ATGATG interruptions were placed at two different positions within the repeating sequence. All sequences are shown as the lagging strand template.
(C) Analysis of contractions for the perfect and interrupted alleles yielded the indicated rate values expressed as events per cell generation × 10–5. Stabilization factors were calculated as the perfect repeat rate divided by the rate for the interrupted alleles.
(D) A model to explain how contraction rates are reduced by interruptions is shown. A description is provided in the text.
Molecular Cell 2000 6, DOI: ( /S (00) )