title: Unusual structures are present in DNA fragments containing super-long Huntingtin CAG repeats.
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.
  
  
  RESULTS
  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
  "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.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Duzdevich
authors.forename: Jinliang
authors.initials: J
authors.lastname: Li
authors.forename: Jhoon
authors.initials: J
authors.lastname: Whang
authors.forename: Hirohide
authors.initials: H
authors.lastname: Takahashi
authors.forename: Kunio
authors.initials: K
authors.lastname: Takeyasu
authors.forename: David T F
authors.initials: DT
authors.lastname: Dryden
authors.forename: A Jennifer
authors.initials: AJ
authors.lastname: Morton
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D005838:Genotype; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics; D055029:Inverted Repeat Sequences / Q000235:genetics; D051379:Mice; D018625:Microscopy, Atomic Force; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D018911:Trinucleotide Repeats / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D004247:DNA; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)