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R loops stimulate genetic instability of CTG.CAG repeats.

Yunfu Lin1, Sharon Y R Dent, John H Wilson

  • 1Baylor College of Medicine, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, One Baylor Plaza, Houston, TX 77030, USA.

Proceedings of the National Academy of Sciences of the United States of America
|January 19, 2010
PubMed
Summary

Transcription-induced genetic instability in trinucleotide repeats is driven by stable RNA:DNA hybrids. Ribonuclease H activity is crucial for preventing this instability, highlighting a key mechanism in repeat sequence variation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Transcription can increase genetic instability in trinucleotide repeat sequences.
  • The precise mechanisms behind transcription-dependent repeat length variation remain poorly understood.

Purpose of the Study:

  • To investigate the role of RNA:DNA hybrids in the genetic instability of CTG.CAG repeat sequences during transcription.
  • To elucidate the mechanisms linking transcription to repeat length variation.

Main Methods:

  • Biochemical assays to assess RNA:DNA hybrid formation and stability.
  • Genetic studies in E. coli and human cells involving mutations and gene knockdown.
  • Ribonuclease H (RNase H) treatment and activity assays.
  • Bisulfite modification to detect single-stranded DNA.

Main Results:

  • Stable RNA:DNA hybrids form readily in CG-rich repeating sequences during transcription, unlike AT-rich or non-repeating sequences.
  • These RNA:DNA hybrids are resistant to RNase A but are degraded by RNase H.
  • Decreased RNase HI activity or RNase H1/H2 knockdown in E. coli and human cells, respectively, significantly enhances CTG.CAG repeat instability, particularly during active transcription.
  • Evidence suggests that RNA:DNA hybrids displace the non-template DNA strand at transcribed CTG.CAG repeats.

Conclusions:

  • Persistent RNA:DNA hybrids formed between nascent RNA transcripts and template DNA strands are a key driver of genetic instability in CTG.CAG trinucleotide repeat tracts.
  • RNase H activity plays a critical role in mitigating this instability by resolving these hybrids.
  • Understanding this mechanism is vital for comprehending repeat expansion disorders.