Abstract
Instability of (CTG)•(CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG)•(CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG)•(CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.
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Acknowledgements
The authors thank C. Pearson and D.G. Monckton for their comments on this work. This work was supported by a grant from the Wright State University Boonshoft School of Medicine to G.L. and by grants from the US National Institutes of Health to M.L. (GM53819) and to J.J.B. (DK61458).
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Experiments were conceived and designed by G.L., M.L., J.J.B. and R.R.S. and performed by G.L., X.C. and M.L. The manuscript was drafted by G.L. and M.L. and revised by all authors.
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Liu, G., Chen, X., Bissler, J. et al. Replication-dependent instability at (CTG)•(CAG) repeat hairpins in human cells. Nat Chem Biol 6, 652–659 (2010). https://doi.org/10.1038/nchembio.416
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DOI: https://doi.org/10.1038/nchembio.416
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