Open AccessR loops stimulate genetic instability of CTG·CAG repeats
Author(s) -
Yunfu Lin,
Sharon Y.R. Dent,
John H. Wilson,
Robert D. Wells,
Марек Напиерала
Publication year - 2009
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0909740107
Subject(s) - biology , rnase h , trinucleotide repeat expansion , transcription (linguistics) , microbiology and biotechnology , genome instability , dna , rnase p , rna , ribonuclease , genetics , gene , dna damage , allele , linguistics , philosophy
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 thernhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG·CAG repeats inE. 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.