Abstract
Expansions of (CAG)n/(CTG)n trinucleotide repeats are responsible for over a dozen neuromuscular and neurodegenerative disorders. Large-scale expansions are commonly observed in human pedigrees and may be explained by iterative small-scale events such as strand slippage during replication or repair DNA synthesis. Alternatively, a distinct mechanism may lead to a large-scale repeat expansion as a single step. To distinguish between these possibilities, we developed a novel experimental system specifically tuned to analyze large-scale expansions of (CAG)n/(CTG)n repeats in Saccharomyces cerevisiae. The median size of repeat expansions was ∼60 triplets, although we also observed additions of more than 150 triplets. Genetic analysis revealed that Rad51, Rad52, Mre11, Pol32, Pif1, and Mus81 and/or Yen1 proteins are required for large-scale expansions, whereas proteins previously implicated in small-scale expansions are not involved. From these results, we propose a new model for large-scale expansions, which is based on the recovery of replication forks broken at (CAG)n/(CTG)n repeats via break-induced replication.
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Acknowledgements
Research in the laboratory of S.M.M. is supported by NIH grants R01GM60987 and P01GM105473 and by a generous contribution from the White family. J.C.K. was supported by the NIH Training in Education and Critical Research Skills postdoctoral program (K12GM074869) and by Tufts University. We thank C. Freudenreich for valuable comments on the manuscript, G. Ira and S. Jinks-Robertson for helpful discussions, K. Spivakovsky-Gonzalez for help analyzing MSH mutants, and D. Padmanhaban for help with strain construction. S.T.H. received support from REU award NSF DBI 1263030. T.D. received support from Tufts Summer Scholars.
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J.C.K. and S.M.M. designed the study; J.C.K., S.T.H., and T.D. performed experiments; J.C.K., S.T.H., T.D., and S.M.M. analyzed data; K.A.S. provided reagents; J.C.K. and S.M.M. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Sequence of the cassette used to generate the (CAG)140 strain YJK146.
Gray letters show chromosome III sequences used to target the construct to chromosome III (SGD coordinates 75,227–75,594 at the beginning of the cassette and coordinates 75,641–75,895 at the end). Sequence from the GAL1 promoter is in orange with the four Gal4 binding sites in yellow (Frolova, E. et al., Nucleic Acids Research. 27, 1350–1358, 1999). Other sequences are from the tetracycline resistance gene (green), CAG repeats (lime), TATA box (magenta), CAN1 (turquoise), and TRP1 (blue). Unmarked sequences are from the vector/plasmid.
Supplementary Figure 2 Construction of the (CAG)140 strain used to analyze large-scale CAG-repeat expansions in Saccharomyces cerevisiae.
Spot assay of strains containing no repeats and (CAG)140 with varying distances from UAS (last Gal4 binding site) to TATA box. Spots show 10-fold serial dilutions. Canavanine plate is shown after 5 days of growth at 30°C. Strain marked with red box (YJK146) was used for expansion rate analysis in various gene knockouts.
Supplementary Figure 3 Large-scale CAG-repeat expansions can be recovered and analyzed in budding yeast.
(A) Experimental system to study large-scale CAG repeat expansions. In the starting strain, the distance between the upstream activating sequence (UASGAL) and TATA box promoter (PGAL) allows transcription of the forward selection marker CAN1, which encodes an arginine permease. Large-scale expansions will result in CanR clones. Bars above the CAG repeats indicate products of single colony PCR used for sequencing (PCR1) and determination of repeat length (PCR2). (B) Agarose gel images showing PCR amplification of CAG repeat length for CanR clones. Arrow points to the initial length of (CAG)140. Only expanded clones are used to calculate expansion rates. Asterisks in lanes 2, 3, 6, and 8 indicate expanded clones. Clone 5 may be a large expansion with secondary contractions or the result of a gross chromosomal rearrangement.
Supplementary Figure 4 Genetic control of large-scale CAG-repeat expansions.
Effect of different gene knockouts and mutations on the large-scale expansion rate of (CAG)140 (A) Non-selective growth on galactose. Selection at 60 μg/mL canavanine concentration (B) Non-selective growth on glucose. Selection at 60 μg/mL canavanine concentration Dashed line designates 3-fold decrease from wild-type. Numbers above the dashed line show fold decrease compared to wild-type. Dotted line in (A) indicates 3-fold increase from wild-type.
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Kim, J., Harris, S., Dinter, T. et al. The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats. Nat Struct Mol Biol 24, 55–60 (2017). https://doi.org/10.1038/nsmb.3334
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DOI: https://doi.org/10.1038/nsmb.3334
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