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 highthroughput 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.

Decrease in topoisomerase I is responsible for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation

“…NTZ-AIDER cells treated with mTop1 #35 or mTop1 #97 showed approximately doubled rates of GFP reversion (0.9% and 0.95%) compared with the control (0.55%) in the absence of tet, whereas the GFP reversion gradually declined, reaching almost zero at 50 ng/mL tet, regardless of the Top1 knockdown. These results clearly indicate that transcription has the dominant effect in SHM, which is also true for TAM, CAG-repeat deletion, and CSR (21,24,25,27).…”

“…Gangloff et al (46) showed that the loss of Top1 increases genetic instability in yeast and suggested that CSR may depend on transcription-induced non-B configuration in the repetitive sequences of the Sα region during CSR. Recently, Top1 was also shown to be involved in the transcription-induced tripletrepeat deletion in mammals (27). Top1 knockdown causes an increase in triplet contraction.…”

“…When the transcription rate exceeds the capacity of Top1, excessive negative supercoiling accumulates at the rear of the transcription machinery, increasing the chance of non-B structure formation at repeat or palindrome-rich regions (29). Top1 cuts non-B DNA and forms a DNA-bound intermediate but cannot rotate around the helix, resulting in irreversible cleavage and mutagenesis (27). Thus, the appropriate balance between the transcription rate and Top1 activity appears to be critical for maintaining genome stability.…”

“…Triplet repeats are believed to form non-B structures caused by the excessive negative superhelicity induced by active transcription. A recent report provided evidence that Top1 introduces irreversible cleavage at triplet repeats in a transcription-dependent manner (27).…”

An Evolutionary View of the Mechanism for Immune and Genome Diversity

“…In addition, in the many triplet-associated diseases, such as Huntington's disease (2), the triplets are usually located within the transcribed region of genes, and the triplet repeats are either expanded or contracted in somatic cells. Recent studies showed that topoisomerase 1 (Top1) is involved in both TAM and the triplet contraction/expansion (3)(4)(5).…”