ALTERNATIVE STRUCTURES IN DUPLEX DNA FORMED WITHIN THE TRINUCLEOTIDE REPEATS OF THE MYOTONIC-DYSTROPHY AND FRAGILE-X LOCI

Most models proposed to explain the disease-associated expansion of (C TG)(n) .(CAG)(n) and (CGG)(n) .(CCG)(n) trinucleotide repeats include the formation of slipped strand DNA structures during replication; how ever, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystr ophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premu tation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repe at tracts during reannealing of complementary strands, containing equa l lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures ar e characterized by reduced electrophoretic mobilities in polyacrylamid e gels. These alternative secondary structures are stable at physiolog ical ionic strengths and to temperatures up to at least 55 degrees C. Following reduplexing, the CAG strand of the (CTG)(n) .(CAG)(n) repeat s is preferentially sensitive to mung bean nuclease, suggesting the pr esence of single-stranded DNA in the alternative DNA structure. For (C TG)(17), which is a repeat length found in normal individuals, less th an 3% of the DNA molecules formed alternative DNA structures upon redu plexing. DNA molecules containing (CTG)(50) or (CTG)(255), which repre sent premutation and full mutation lengths of triplet repeats, respect ively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed inc reased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the n ovel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential in volvement of these structures, which we term S-DNA, in the genetic ins tability of triplet repeats is discussed.