Slipped-strand DNAs formed by long (CAG)middle dot(CTG) repeats: slipped-out repeats and slip-out junctions

Abstract

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.

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…Mung bean nuclease is also highly sensitive to variations in DNA structure and converts single-stranded or unpaired DNA to mono-or oligonucleotides with 5Ј-phosphates (82)(83)(84)98). The probing of d(CAGG) 26 with mung bean nuclease showed predominant cleavage between the first and second guanines of the 13th CAGG repeat, the cytosine and adenine, as well as the first and second guanine residues of the 14th CAGG repeat.…”

Fidelity of Primate Cell Repair of a Double-strand Break within a (CTG)·(CAG) Tract

“…6). We have recently demonstrated that both S-DNA and SI-DNA structures can be specifically recognized and cleaved by both junction-and single strand-specific nucleases (36). Mammalian cells are believed to use non-homologous end joining (NHEJ) as a primary means of DSB repair (26), making proteins such as Ku70, Ku80, DNA-PKcs, DNA ligase IV, XRCC4, and Artemis likely candidates.…”

Mismatch Recognition Protein MutSβ Does Not Hijack (CAG) Hairpin Repair in Vitro

“…The precise mechanisms by which TNR expansion occurs and the factors that promote it are not fully understood. It has been proposed that CAG and CTG repeats form thermostable hairpins that include A-A and T-T mispairs in the hairpin stem (4,5). Therefore, cellular mechanisms that process DNA hairpin/loop structures and/or A-A or T-T mispairs may influence TNR stability.…”