around 4 months to over 18 months in mice with the longest repeats [17]. Increased repeat length was associated with a reduced nuclear entry of the larger htt fragments [18], which will cause an amelioration of the R6/2 phenotype relating to the formation of nuclear inclusions [44,45,46]. A reduced transcrip- tion of the transgene was also seen at long repeat lengths, which is known to be associated with an amelioration of the R6/2 phenotype [16,47]. We speculate that the generation of unusual DNA structures seen in our in vitro experiments might underlie the observed reduction in transcription observed in vivo. Significantly, it has been shown previously that Hdh mRNA levels are reduced in knock-in mice containing 150 CAG repeats [19]. Consistent with this observation, we saw fewer of the unusual structures in short repeat length DNA, but they were still present. It is not clear if there is a reduced level of mutant htt in mice with superlong CAG repeat lengths. We know that htt with superlong CAG repeats is translated, because bands of the appropriate size can be visualized using anti-htt antibodies when we run proteins from the allelic series of mice on immunoblots (data not shown). However, because mutant htt forms aggregates, accurate protein quantita- tion is not possible (for detailed discussion of this issue, see [17]). In addition to the insights provided into the HD phenotype, our study also sheds light on the mechanism of action of two very different restriction enzymes. The observation that at 80uC Tse1 cleaves within the hairpins indicates that an A:A or T:T mismatch at the centre of the recognition sequence (59-GC(A/T)GC-39) does not prevent its restrictive activity. Tse1 may therefore belong to an interesting group of restriction enzymes which flip out nucleotides within a recognition sequence (e.g. [48]). In the few studied cases, the restriction enzyme uses a flipping mechanism to recognize a sequence or adjust the shape of the DNA within its catalytic site for proper cleavage. Either of these scenarios might occur when Tse1 encounters dsDNA. We suggest that nucleotide flipping may allow it to recognize and cleave near an A:A or T:T mismatch, or to collapse the recognition sequence to 59-GCGC-39, thereby ignoring mismatches. These possibilities remain to be tested. The ability of EcoP15I to cleave the convoluted structures selectively is a particularly interesting phenomenon, given that two head-to-head recognition sites are normally required for its restriction activity [37,38,49]. Our data suggest that the EcoP15I enzyme may be recognizing the sequence 59-CXGCXG-39 where X represents the A:A or T:T mismatch. Significantly, a previous study of EcoP15I activity using 2-aminopurine substitutions within the cognate sequence [50] revealed distortion (probably base- flipping) at the two adenine sites upon enzyme binding. The same study also showed preferential binding to target sequences containing a mismatch. Preceding each hairpin will be perfect repeats containing the normal EcoP15I target sequence, 59- CAGCAG-39, which it would appear can interact with the mismatch sequence to produce cleavage. Further investigation of the action of these restriction enzymes on CAG-repeat DNA is clearly warranted. With respect to the results presented here, the central question is: could the generation of unusual DNA structures play a role in the aetiology of HD? It is certainly clear that CAG repeats in the appropriate range exist in HD patients [9]. It is also known that the production of hairpin loops in triplet repeat DNA is a potential mechanism underlying repeat instability, which is a feature of triplet repeat disorders such as HD [2,20,51,52]. It is not known, however, if abnormal secondary structures exist in DNA in vivo. The DNA imaged in our study was generated by PCR, which of course uses thermal cycling to drive melting and reannealing. However, a similar cycling between double- and single-stranded Unusual Structures in Super-Long CAG Repeat DNA DNA states occurs during the processes of transcription, replication and recombination, suggesting that unusual DNA structures might also be generated in vivo. The observation that mRNA levels are reduced in HD mouse models with super-long CAG repeats supports this suggestion. While CAG repeat disorders such as HD typically have a relatively short (,80) CAG repeat that causes disease, there are several studies showing that CAG repeat instability can generate CAG repeat lengths of greater than 300 in individual cells [10,13]. There are also a large number of diseases, including myotonic dystrophy type 1 and Fragile X syndrome, where the disease- causing mutations consist of repeats (in non-coding regions of the genome) that are well above the size of repeats found here to generate unusual DNA structures. We speculate that if the conformational changes in DNA that we have described occur in vivo, they may contribute to pathology associated with trinucleotide repeat diseases. They could do this by allowing a gain of function, for example, by permitting cleavage by enzymes that normally do not cut, or by preventing binding of regulatory proteins to dysregulate transcription. They may also contribute to alterations in chromatin structure that are known to occur in CAG repeat diseases [53]. It is unlikely that the reduction of transcription causes the disease phenotype, given that only one functional allele of HTT is needed and that mutant HTT can rescue the lethality in mutants. But it is also possible that the presence of super-long CAG repeat expansions may have a paradoxically ‘protective’ effect. For instance, by reducing transcription of the mutant DNA, they may reduce the amount of toxic mutant protein, and delay the onset of disease. However, these ideas must be regarded as speculative, since at present it is not possible to determine whether or not the abnormal DNA structures that we describe exist in vivo. Supporting Information Figure S1 Observation of convoluted DNA structures does not depend on the presence of Mg2+ in the deposition buffer. DNA with 360 CAG repeats (total length 1211 bp) was deposited on mica that had been treated with either Mg2+ (A) or poly-L-lysine (B). Despite the absence of Mg2+ ions, the DNA bound via poly-L-lysine still exhibited anomalous, non- linear conformations. Scale bars, 500 nm. (TIF) Figure S2 Human s-1 receptor DNA shows no unusual structures. (A) AFM image of undigested s-1 receptor DNA. Note that the DNA is normal and linear. Scale bar, 250 nm. (B) s-1 receptor after complete Tse1 digestion at 80uC. Fragments of various sizes are visible after digestion, as expected since the s-1 receptor sequence contains five TseI cleavage sites. Scale bar, 500 nm. (TIF) Acknowledgments We are grateful to Dr. Robert Henderson (Department of Pharmacology, University of Cambridge) for helpful discussions. Author Contributions Conceived and designed the experiments: DD KT DTFD AJM JME. Performed the experiments: DD JL JW HT. Analyzed the data: DD JL HT DTFD AJM JME. Contributed reagents/materials/analysis tools: JL JW. Wrote the paper: DD KT DTFD AJM JME. PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e17119