Topoisomerase 1 and Single-Strand Break Repair Modulate Transcription-Induced CAG Repeat Contraction in Human Cells

Abstract

Expanded trinucleotide repeats are responsible for a number of neurodegenerative diseases, such as Huntington disease and myotonic dystrophy type 1. The mechanisms that underlie repeat instability in the germ line and in the somatic tissues of human patients are undefined. Using a selection assay based on contraction of CAG repeat tracts in human cells, we screened the Prestwick chemical library in a moderately high-throughput assay and identified 18 novel inducers of repeat contraction. A subset of these compounds targeted pathways involved in the management of DNA supercoiling associated with transcription. Further analyses using both small molecule inhibitors and small interfering RNA (siRNA)-mediated knockdowns demonstrated the involvement of topoisomerase 1 (TOP1), tyrosyl-DNA phosphodiesterase 1 (TDP1), and single-strand break repair (SSBR) in modulating transcription-dependent CAG repeat contractions. The TOP1-TDP1-SSBR pathway normally functions to suppress repeat instability, since interfering with it stimulated repeat contractions. We further showed that the increase in repeat contractions when the TOP1-TDP1-SSBR pathway is compromised arises via transcription-coupled nucleotide excision repair, a previously identified contributor to transcription-induced repeat instability. These studies broaden the scope of pathways involved in transcription-induced CAG repeat instability and begin to define their interrelationships.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.05158-11.

ACKNOWLEDGMENTS

We thank Zaowen Chen and Jason Shohet for help with real-time RT-PCR. We thank Michele Washington for characterization of the siRNA against PARP1 and the Western blot to determine PARP1 knockdown in cells. We thank Motonari Uesugi for advice and help with the Prestwick chemical library. We thank members of the Wilson lab for helpful discussions.

This study was supported by NIH grant 1F31HG004918 to L.H., Natural Sciences and Engineering Research Council of Canada postgraduate scholarship D to V.D., and NIH grant GM38219 to J.H.W.