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Slipped (CTG)•(CAG) repeats can be correctly repaired, escape repair or undergo error-prone repair

Abstract

Expansion of (CTG)•(CAG) repeats, the cause of 14 or more diseases, is presumed to arise through escaped repair of slipped DNAs. We report the fidelity of slipped-DNA repair using human cell extracts and DNAs with slip-outs of (CAG)20 or (CTG)20. Three outcomes occurred: correct repair, escaped repair and error-prone repair. The choice of repair path depended on nick location and slip-out composition (CAG or CTG). A new form of error-prone repair was detected whereby excess repeats were incompletely excised, constituting a previously unknown path to generate expansions but not deletions. Neuron-like cell extracts yielded each of the three repair outcomes, supporting a role for these processes in (CTG)•(CAG) instability in patient post-mitotic brain cells. Mismatch repair (MMR) and nucleotide excision repair (NER) proteins hMSH2, hMSH3, hMLH1, XPF, XPG or polymerase β were not required—indicating that their role in instability may precede that of slip-out processing. Differential processing of slipped repeats may explain the differences in mutation patterns between various disease loci or tissues.

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Figure 1: Slipped-strand intermediates during replication, repair and recombination.
Figure 2: Slipped-strand intermediate substrates.
Figure 3: In vitro slipped-strand repair.
Figure 4: Substrate-specific factors affect slipped-strand repair.
Figure 5: Repair efficiencies using repair-proficient and repair-deficient cell extracts with a possible processing mechanism.
Figure 6: A proposed mechanism for correct, escaped or error-prone repair of slipped-DNA intermediates, with newly incorporated regions of specific and nonspecific synthesis indicated by dots.

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Acknowledgements

We thank J. Danska, C.J. Ingles, A. La Spada, M.S. Meyn, T.D. Petes and H.Y. Zoghbi for comments on the manuscript, P. Modrich for the G•T substrate, C.R. Boland for the HCT116 cells, S. Påhlman for tips on differentiation of SH-SY5Y, A. Todd and K. Nichol Edamura for technical and experimental support, and members of the Pearson lab for intellectual support. We also acknowledge our debt to Arthur Kornberg for Commandment VIII (“Respect the personality of DNA”) of his Ten Commandments of DNA, in Biology of DNA (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003). Our work here was supported by the Canadian Institutes of Health Research (CIHR) and The Muscular Dystrophy Association (USA). S.E.M. and M.L.R. were supported by a Research Training Studentship (The Hospital for Sick Children) and Ontario Graduate Scholarships. G.B.P. was supported by a Premier's Research Excellence Award to C.E.P. C.E.P. is a CIHR Scholar and a Canadian Genetic Disease Scholar.

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Correspondence to Christopher E Pearson.

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Supplementary information

Supplementary Fig. 1

Preparation of circular slipped-heteroduplexes. (PDF 84 kb)

Supplementary Fig. 2

Biophysically characterized structures of substrates used by Pearson Lab. (PDF 116 kb)

Supplementary Fig. 3

In vitro G-T mismatch repair. (PDF 241 kb)

Supplementary Fig. 4

Repair is structure-dependent. (PDF 147 kb)

Supplementary Fig. 5

Repair incorporation mapped to the repeat-containing fragment and was predominantly to the nicked strand. (PDF 727 kb)

Supplementary Fig. 6

Ladder band products were a series of SI-DNAs. (PDF 854 kb)

Supplementary Fig. 7

Genetic background of the cells used. (PDF 38 kb)

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Panigrahi, G., Lau, R., Montgomery, S. et al. Slipped (CTG)•(CAG) repeats can be correctly repaired, escape repair or undergo error-prone repair. Nat Struct Mol Biol 12, 654–662 (2005). https://doi.org/10.1038/nsmb959

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