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Nucleic Acids Research, Vol 27, Issue 13 2610-2617, Copyright © 1999 by Oxford University Press

ARTICLES

DNA structural transitions within the PKD1 gene

RT Blaszak, V Potaman, RR Sinden and JJ Bissler
The Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single- stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H- y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.
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