Sticky DNA: in Vivo Formation in E. coli and in Vitro Association of Long GAA•TTC Tracts to Generate Two Independent Supercoiled Domains

Abstract

The expanded GAA•TTC repeat sequence associated with Friedreich’s ataxia (FRDA) adopts non- B DNA structures, (triplexes and sticky DNA). Sticky DNA is formed in plasmids by the association of two long GAA•TTC tracts at lengths that are found in the sequence of the frataxin gene in patients. Most FRDA patients have expanded GAA•TTC repeats (up to 1700 triplets), which inhibit the transcription of the gene, thus diminishing the synthesis of frataxin, a mitochondrial protein involved in iron-sulfur cluster biogenesis. Negative supercoiling and MgCl 2 (or MnCl 2) are required to stabilize sticky DNA (a dumbbell-shaped structure) in plasmids with a pair of repeat tracts where n ≥ 60 in the direct repeat orientation in vitro. Since the triplet repeat sequences (TRS) were symmetrically positioned in the plasmids and because a number of unique restriction sites were present in the vector, studies were conducted to evaluate the influence of selectively linearizing one or the other supercoiled domains created by the DNA•DNA associated region, i.e. the stable complex at the pair of TRS’s. The two domains behave independently, thus confirming the association of the two tracts and the dumbbell-shaped plasmid in our model for sticky DNA. Linking number investigations were performed on a family of plasmids harboring different lengths (30, 60, or 176 repeats), orientations and number of tracts (one or two) of a GAA•TTC repeat in Escherichia coli to evaluate the in vivo role, if any, of sticky DNA. Unexpectedly, this non- B DNA conformation elicited the formation of a TRS-length dependent change in the global topology of the plasmids, indicative of an apparent compression of the primary helices. Thus, linking number determinations confirm that sticky DNA has an important consequence in vivo.