Unpaired Structures in SCA10 (ATTCT) n ·(AGAAT) n Repeats Vladimir N. Potaman 1 , John J. Bissler 2 , Vera I. Hashem 1 Elena A. Oussatcheva 1 , Lu Lu 2 , Luda S. Shlyakhtenko 3 Yuri L. Lyubchenko 3 , Tohru Matsuura 4 , Tetsuo Ashizawa 5 Michael Leffak 6 , Craig J. Benham 7 and Richard R. Sinden 1 * 1 Laboratory of DNA Structure and Function, Center for Genome Research, Institute of Biosciences and Technology Texas A and M University System Health Sciences Center Houston, TX 77030-3303 USA 2 Division of Nephrology and Hypertension, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA 3 Departments of Biology and Microbiology, Arizona State University, Tempe, AZ 85287-2701, USA 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston TX 77030, USA 5 Department of Neurology, The University of Texas Medical Branch, Galveston, TX 77555-0539, USA 6 Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435-0001, USA 7 Department of Mathematics University of California, Davis Davis, CA 95616-8633, USA A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT) n ·(AGAAT) n from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electro- phoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT) n ·(AGAAT) n repeat forms an unpaired region, which further extends into adjacent A þ T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT) 5 ·(AGAAT) 5 occurred while 2D gel analysis and chemical probing indicate greater unpairing in A þ T-rich sequences in other regions of the plasmid. The observed experimental results are con- sistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A þ T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high super- helical densities, DNA strands within the presumably compact A þ T-rich region were accessible to small chemicals and oligonucleotide hybridiz- ation. Thus, DNA strands in this “collapsed structure” remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation. q 2003 Elsevier Science Ltd. All rights reserved Keywords: pentanucleotide repeats; DNA supercoiling; DNA unpairing; DNA replication initiation; DNA unwinding element *Corresponding author Introduction Eighteen human genetic diseases have been associated with the expansion of DNA repeats. Diseases associated with non-coding (CGG) n · (CCG) n triplet repeats include fragile X syndrome 0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: rsinden@ibt.tamu.edu Abbreviations used: SCA10, spinocerebellar ataxia type 10; CAA, chloroacetaldehyde; DUE, DNA unwinding element; BUR, base unpairing region; AFM, atomic force microscopy. doi:10.1016/S0022-2836(03)00037-8 J. Mol. Biol. (2003) 326, 1095–1111