A GENOME-DERIVED (GAA.TTC)(24) TRINUCLEOTIDE BLOCK BINDS NUCLEAR PROTEIN(S) SPECIFICALLY AND FORMS TRIPLE HELICES

The properties of simple trinucleotide repeats generate increased inte rest as expansions of certain trinucleotide blocks cause human disease s. Here, we studied protein binding and structural features of a perfe ct (gaa.ttc)(24) tract in its original genomic environment. Electropho retic mobility shift assays revealed that HeLa nuclear proteins bind t o the DNA fragment containing the (gaa.ttc)(24) block. Competition exp eriments using simple (gt.ac)(n) repeats differing in length and flank ing regions showed no crossreactivity with the major retarded band. Fo r the specific (gaa.ttc)(n)/protein complex, a binding constant of 9.3 x 10(-9) mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)(24) strand. OsO4 and DEPC modifications followed by elec trophoretic and electron microscopical analyses showed that the (gaa.t tc)(24) block forms different types of intramolecular triple helices: Under superhelical stress, different H-DNA isomers are evident, where as exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)(n) trac ts. (C) 1998 Elsevier Science B.V. All rights reserved.