Abstract
R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.
Highlights
R-loops are thermodynamically stable, RNA:DNA structures that may form during transcription
To investigate the role of R-loops in (CAG)·(CTG) repeat instability, we utilized human whole cell extracts (HeLa) that are proficient in in vitro DNA repair in an in vitro processing assay which we modified from previous studies of repeat instability in the lab [26,31,34,35]
We show that R-loops generated by in vitro transcription of an expanded (CAG)79·(CTG)79 DNA repeat tract as well as an expanded (GGGGCC)40·(GGCCCC)40 can be processed by human cell extract to increase instability of the repeat tract
Summary
R-loops are thermodynamically stable, RNA:DNA structures that may form during transcription. Biological roles for R-loops have been described in DNA replication initiation at mitochondrial and prokaryotic origins of replication, in class switch recombination at immunoglobulin genes, and in DNA methylation regulation at CpG island sequences [4,5,6,7]. In these cases, the formation of R-loops is a necessary event regulating essential downstream activities. Mutations in the THO/TREX complex, which is required for proper coupling of transcription and mRNA export, cause wide-scale co-transcriptional R-loop formation triggering aberrant recombination leading to genome-wide instability [8,9,10]. Gene-specific R-loop-mediated mutagenesis may have specific sequence requirements, as described below
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