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Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system

Nature Structural & Molecular Biology volume 25, pages 669–676 (2018)Cite this article

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Abstract

We developed an experimental system for studying genome instability caused by fragile X (CGG)n repeats in mammalian cells. Our method uses a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)n repeats in its 5′ UTR, which is integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)n repeats markedly elevated the frequency of reporter inactivation, making cells ganciclovir resistant. These resistant clones had a unique mutational signature: a change in repeat length concurrent with mutagenesis in the reporter gene. Inactivation of genes implicated in break-induced replication, including Pold3, Pold4, Rad52, Rad51, and Smarcal1, reduced the frequency of ganciclovir-resistant clones to the baseline level that was observed in the absence of (CGG)n repeats. We propose that replication fork collapse at carrier-size (CGG)n repeats can trigger break-induced replication, which results in simultaneous repeat length changes and mutagenesis at a distance.

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Fig. 1: Experimental system to study genome instability caused by (CGG)n repeats.
Fig. 2: Mechanisms of ganciclovir resistance.
Fig. 3: Proposed mechanism for mutational events triggered by carrier-size (CGG)n repeats.

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Acknowledgements

We thank K. Usdin (NIDDK, NIH) for the generous gift of the p32.9 plasmid, E. Bouhassira (Albert Einstein College of Medicine) for providing us with the RL5 cell line, D. Gennert for technical assistance, and A. Neil for his invaluable editorial help. This work was supported by NIH grants R01GM60987 and P01GM105473 to S.M.M. and R01CA093729 to K.M.V.

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  1. These authors contributed equally: Artem V. Kononenko, Thomas Ebersole.

Authors and Affiliations

  1. Department of Biology, Tufts University, Medford, MA, USA

    Artem V. Kononenko, Thomas Ebersole & Sergei M. Mirkin

  2. Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin, TX, USA

    Karen M. Vasquez

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Contributions

A.V.K., T.E., K.M.V. and S.M.M. designed the study; A.V.K. and T.E. performed experiments; A.V.K., T.E. and S.M.M. analyzed data; A.V.K., T.E., K.M.V. and S.M.M. wrote the manuscript.

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Correspondence to Sergei M. Mirkin.

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Supplementary Figure 1 Mutations found in the TK domain of the HyTK gene in various FMR1-(CGG)n-HyTK cassettes.

DNA bases in the TK domain of the wild-type HyTK gene that underwent mutagenesis are shown in bold. Mutations found in the cassettes with (CGG)0, (CGG)53, and (CGG)153 repeats are shown in green, blue, and red, respectively; mutations found in the cassette with (CGG)153 repeats upon treatment with Pold3 siRNA are shown in orange; and G7 and C6 mutation hotspots are highlighted in yellow. V, insertion; Δ, deletion; D, duplication; FV, insertion to frameshift; FΔ, deletion to frameshift; X, complex mutation; AT and TT, tandem base substitutions.

Supplementary Figure 2 PCR analysis of repeat lengths for clones originated after treatment of the FMR1-(CGG)153-HyTK cell line with Pold3 siRNA.

Lane 1 shows the original repeat; lanes 2–4, 6, 8, and 9 show expanded repeats; and lanes 5 and 7 show unchanged repeat. Mutations found within the HyTK gene are shown at the top of the corresponsing lane.

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Supplementary Text and Figures

Supplementary Figures 1 and 2, and Supplementary Table 1

Reporting Summary

Supplementary Dataset 1

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Kononenko, A.V., Ebersole, T., Vasquez, K.M. et al. Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system. Nat Struct Mol Biol 25, 669–676 (2018). https://doi.org/10.1038/s41594-018-0094-9

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