Slipped‐strand DNAs formed by long (CAG)·(CTG) repeats: slipped‐out repeats and slip‐out junctions

摘要

The disease‐associated expansion of (CTG)·(CAG) repeats is likely to involve slipped‐strand DNAs. There are two types of slipped DNAs (S‐DNAs): slipped homoduplex S‐DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI‐DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S‐DNAs formed by disease‐relevant lengths of (CTG)·(CAG) repeats which contained all predicted components including slipped‐out repeats and slip‐out junctions, where two arms of the three‐way junction were composed of complementary paired repeats. In S‐DNAs multiple short slip‐outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI‐DNAs most of the excess repeats slipped‐out at preferred locations along the fully base‐paired Watson–Crick duplex, forming defined three‐way slip‐out junctions. Unexpectedly, slipped‐out CAG and slipped‐out CTG repeats were predominantly in the random‐coil and hairpin conformations, respectively. Both the junctions and the slip‐outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction‐specific T7 endonuclease I, while slipped‐out CAG was preferentially bound by single‐strand binding protein. An excellent correlation was observed for the size of the slip‐outs in S‐DNAs and SI‐DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients—suggesting that S‐DNAs and SI‐DNAs are mutagenic intermediates in those tissues, occurring during error‐prone DNA metabolism and replication fork errors.