Slipped-strand DNAs formed by long (CAG)*(CTG) repeats: slipped-out repeats and slip-out junctions.
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.
Pearson
Christopher E
CE
Program of Genetics and Genomic Biology, The Hospital for Sick Children, 555 University Avenue, Elm Wing 11-135, Toronto, Ontario M5G 1X8, Canada. cepearson@genet.sickkids.on.ca
Tam
Mandy
M
Wang
Yuh-Hwa
YH
Montgomery
S Erin
SE
Dar
Arvin C
AC
Cleary
John D
JD
Nichol
Kerrie
K
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012091:Repetitive Sequences, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific; C025629:GCAGC-specific type II deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases