Oligodeoxynucleotide binding to (CTG) · (CAG) microsatellite repeats inhibits replication fork stalling, hairpin...

(CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.