STRUCTURAL-ANALYSIS OF SLIPPED-STRAND DNA (S-DNA) FORMED IN (CTG)(N)CENTER-DOT(CAG)(N) REPEATS FROM THE MYOTONIC-DYSTROPHY LOCUS

The mechanism of disease-associated trinucleotide repeat length variat ion may involve slippage of the triplet-containing strand at the repli cation fork, generating a slipped-strand DNA structure. We recently re ported formation in vitro of slipped-strand DNA (S-DNA) structures whe n DNAs containing triplet repeat blocks of myotonic dystrophy or fragi le X diseases were melted and allowed to reanneal to form duplexes. He re additional evidence is presented that is consistent with the existe nce of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats . in addition, we skew that both the propensity for S-DNA formation an d the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microsc opy, confirming that the two strands are slipped out of register With respect to each other and confirming the structural polymorphism expec ted within long tracts of trinucleotide repeats. For (CTG)(50).(CAG)(5 0) two distinct populations of slipped structures have been identified : those involving less than or equal to 10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternati ve DNA secondary structures.