Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

Daniel Duzdevich, Jinliang Li, Jhoon Whang, Hirohide Takahashi, Kunio Takeyasu, David T. F. Dryden, A. Jennifer Morton, J. Michael Edwardson

Research output: Contribution to journalArticlepeer-review

Abstract

Background: In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.

Methodology/Principal Findings: We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I.

Conclusions/Significance: "Super-long'' CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD.

Original languageEnglish
Article numbere17119
Pages (from-to)-
Number of pages12
JournalPLoS ONE
Volume6
Issue number2
DOIs
Publication statusPublished - 11 Feb 2011

Cite this