R loops stimulate genetic instability of CTG·CAG repeats

Yunfu Lin, Sharon Y.R. Dent, John H. Wilson, Robert D. Wells, Marek Napierala

Research output: Contribution to journalArticlepeer-review

129 Scopus citations

Abstract

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcription-dependent 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.

Original languageEnglish (US)
Pages (from-to)692-697
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number2
DOIs
StatePublished - 2010
Externally publishedYes

Keywords

  • RNA·DNA hybrids
  • Transcription-induced instability
  • Triplet repeats

ASJC Scopus subject areas

  • General

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