R loops stimulate genetic instability of CTG·CAG repeats

Abstract

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcriptiondependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA · DNA hybrids enhances the instability of CTG · CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA · DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG · CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG · CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG · CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA · DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG · CAG tracts promote instability of DNA trinucleotide repeats.RNA·DNA hybrids | transcription-induced instability | triplet repeats E xpansions of simple repeating sequences (microsatellites) are responsible for more than 20 human diseases (1). Moreover, instability of simple repeats (expansion as well as contraction) is observed throughout the genomes of all organisms studied, and it is considered an important source of genetic variation (2). The molecular basis of this unusual mutation mechanism has been studied extensively in the past 15 years using bacteria, yeast, flies, mice, and mammalian cells. Although these analyses uncovered several cis elements and trans-acting factors affecting repeat instability (3), a unifying, comprehensive model of the repeat expansion and contraction is lacking. Processes such as replication, recombination, and repair, which temporarily dissociate DNA complementary strands, strongly destabilize repeating sequences. Transient exposure of single-stranded DNA regions allows formation of non-B DNA structures in regions containing repeats. Virtually all current models of repeat instability incorporate non-B DNA structures as the proximate cause of instability (4, 5).Recently, transcription through tandem repeat sequences has emerged as an important factor promoting instability of repeat sequences (6-12). Although the synthesis of RNA does not change the length of a DNA template, it can lead to the formation of non-B DNA structures, as shown in E. coli, yeast, and higher organisms (7-10, 13). These secondary structures may interfere with the progress of RNA polymerase, calling into play various DNA repair processes, whose action leads to repeat expansion and contraction. In h...

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

“…There is generally good agreement between results in our assay in human cells and those in mouse models (29)(30)(31). Among the 20 genes shown to affect CAG repeat contraction in human cells (6,27,28,31,32,37), six have been tested in a mouse model and also shown to modulate CAG repeat instability, including MSH2 (22,47,48), MSH3 (52), PMS2 (12), DNMT1 (6), CSB (20), and XPA (L. Hubert et al, unpublished data). Similarly, among those genes with little effect on CAG contraction in human cells (28), three have been tested in mouse models and shown to have little effect on CAG repeats, including MSH6 (52), XPC (8), and FEN1 (50,51).…”

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Emerging role of RNA•DNA hybrids inC9orf72-linked neurodegeneration

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