Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats Daniel Duzdevich1, Jinliang Li2, Jhoon Whang 2 , Hirohide Takahashi3, Kunio Takeyasu3, David T. F. Dryden4, A. Jennifer Morton1*, J. Michael Edwardson 1 * 1 Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom, 2 Laragen, Inc., Culver City, California, United States of America, 3 Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, Japan, 4 EaStCHEM School of Chemistry, University of Edinburgh, The King’s Buildings, Edinburgh, United Kingdom 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. Citation: Duzdevich D, Li J, Whang J, Takahashi H, Takeyasu K, et al. (2011) Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats. PLoS ONE 6(2): e17119. doi:10.1371/journal.pone.0017119 Editor: Josef Priller, Charite´-Universita¨tsmedizin Berlin, Germany Received September 28, 2010; Accepted January 21, 2011; Published February 11, 2011 Copyright: 2011 Duzdevich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by CHDI Foundation, Inc. (AJM), a BBSRC Japan Partnering Award (JME), a JSPS Japan-UK Bilateral Joint Project Award (KT), and a JSPS Grant-in-Aid for Basic Research (KT). DD received funding from the Cambridge European Trust. HT holds a Grant-in-Aid Fellowship from the JSPS. DTFD gratefully acknowledges the award of a Derek Brewer Visiting Fellowship at Emmanuel College, Cambridge, during the tenure of which this paper was written. JL and JW are employees of Laragen, Inc., and contributed reagents for the study. The other funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: JJ and JW are employees of Laragen, Inc., a DNA sequencing company. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: ajm41@cam.ac.uk (AJM); jme1000@cam.ac.uk (JME) Introduction At least 30 genetic neurodegenerative diseases involve the expansion of specific DNA triplet repeat sequences [1,2]. These include Fragile X syndrome (CGG repeat), Friedreich’s ataxia (GAA repeat), myotonic dystrophy type I (CTG repeat), and Huntington’s disease (HD; CAG repeat). Depending on the disease, the repeat sequence can be located at various positions relative to the coding region of the gene. Hence, the role of the repeat in the aetiology of the disease will vary. For instance, the CGG repeat diseases appear to involve methylation of the DNA containing the repeats, leading to gene silencing. In contrast, the presence of an expanded CAG repeat in the coding region of HTT, the gene encoding Huntingtin (htt) leads to the generation of an expanded polyglutamine tract in the expressed protein, which produces neurotoxic effects through a gain of function [1]. As the HD-associated CAG repeat expansion is exon-localized, its possible role in controlling gene expression has been largely overlooked. The threshold CAG repeat length that causes HD is around 36, and there is an approximate correlation between the age-at-onset of the disease and the length of the CAG tract [3,4,5]. Patients with 40–42 repeats tend to get the disease in mid-life (35–40 years), whereas patients with more than 60 repeats are afflicted as juveniles [1]. One factor that might blur the relationship between age-at-onset and repeat length is instability of the repeat length, which is known to occur both in humans [6,7,8,9,10] and in mouse models of HD [11,12,13,14,15]. Indeed, ‘super-long’ expansions of CAG repeats up to 1000 have been found in neurons from the brains of patients with adult-onset HD, despite much shorter (41–51) inherited CAG repeats being measured in blood analyses [9,13]. To assess the functional significance of such super-long repeats, R6/2 mice carrying the first exon of the human HD gene with a PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17119