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.