Transcription destabilizes triplet repeats

Abstract

Triplet repeat expansion is the molecular basis for several human diseases. Intensive studies using systems in bacteria, yeast, flies, mammalian cells, and mice have provided important insights into the molecular processes that are responsible for mediating repeat instability. The age-dependent, ongoing repeat instability in somatic tissues, especially in terminally differentiated neurons, strongly suggests a robust role for pathways that are independent of DNA replication. Several genetic studies have indicated that transcription can play a critical role in repeat instability, potentially providing a basis for the instability observed in neurons. Transcription-induced repeat instability can be modulated by several DNA repair proteins, including those involved in mismatch repair (MMR) and transcription-coupled nucleotide excision repair (TC-NER). Though the mechanism is unclear, it is likely that transcription facilitates the formation of repeat-specific secondary structures, which act as intermediates to trigger DNA repair, eventually leading to changes in the length of the repeat tract. In addition, other processes associated with transcription can also modulate repeat instability, as shown in a variety of different systems. Overall, the mechanisms underlying repeat instability in humans are unexpectedly complicated. Because repeat-disease genes are widely expressed, transcription undoubtedly contributes to the repeat instability observed in many diseases, but it may be especially important in nondividing cells. Transcription-induced instability is likely to involve an extensive interplay not only of the core transcription machinery and DNA repair proteins, but also of proteins involved in chromatin remodeling, regulation of supercoiling, and removal of stalled RNA polymerases, as well as local DNA sequence effects.

Figure 1

Substrates used for studying transcription-induced repeat instability in bacteria, yeast, flies, and human cells. In bacteria, transcription is turned on by addition of the inducer, IPTG. Changes in tract length are followed unselectively by gel analysis. In yeast and flies, transcription is turned on by addition of galactose. For yeast, the use of a dinucleotide repeat in the coding sequence shifts the reading frame so that the Ura3 composed of the fusion gene cannot be made. Expansions or contractions of the repeat restore the reading frame and permit cell survival under selection. In flies, changes in tract length are followed unselectively by PCR. In human cells, the transcription is turned on by addition of doxycycline. CAG contractions are followed by HAT selection, which kills HPRT⁻ cells.

Figure 2

Speculative model for transcription-induced repeat instability. In this model, transcription is from left to right and the repeat is oriented so that CTG is on the template (bottom) strand and CAG is on the nontemplate (top) strand. The passage of RNAPII induces secondary structures to form in the repeat tract. Because of their lower thermal stability, CAG structures are shown as loops, while the more stable CTG structures are shown as hairpins. The higher single-strand character of CAG loops would allow them to branch-migrate, permitting multiple smaller loops to coalesce into a larger loop. Stabilization of a CTG hairpin by MutSβ stalls the next RNAPII, which triggers TC-NER. Displacement of RNAPII allows NER proteins to gain access to the hairpin and initiate repair. As shown by the three different pathways, the particular arrangement of the CAG loops versus the CTG hairpins could give rise to three different outcomes: contraction, no change, and expansion of the bottom strand. Remaining structures on the template strand may be repaired by this mechanism, as well. Remaining structures on the nontemplate strand may involve other mechanisms.