Unusual structures are present in DNA fragments containing super-long Huntingtin CAG repeats.

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

Restriction endonuclease TseI cleaves A:A and T:T mismatches in CAG and CTG repeats.

The type II restriction endonuclease TseI recognizes the DNA target sequence 5'-G^CWGC-3' (where W = A or T) and cleaves after the first G to produce fragments with three-base 5'-overhangs. We have determined that it is a dimeric protein capable of cleaving not only its target sequence but also one containing A:A or T:T mismatches at the central base pair in the target sequence. The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair. The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences. The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats)

Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics