Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies.
At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x<y. S-DNAs formed many different species of slipped isomers, as indicated by its multiple electrophoretic species. In contrast, SI-DNAs formed distinct structures, as indicated by the limited electrophoretic species for all possible repeat length pairings. Sister SI-DNAs with an excess of CAG repeats always migrated slower than their sister SI-DNAs with an excess of CTG repeats. Strikingly, both the propensity to form slipped structures and the pattern of S-DNAs, but not SI-DNAs, varied for similar lengths of CTG/CAG repeats between the DM1 and SCA1 loci, highlighting a role for flanking cis-elements in S-DNA but not SI-DNA formation. Slipped structures bound structure and nucleotide-specific anti-DNA antibodies. Binding of anti-B-DNA antibodies was reduced for both S-DNAs and SI-DNAs relative to their linear forms. SI-DNAs bound anti-Z-DNA antibodies, while both S and SI-DNAs bound anti-cruciform antibodies, revealing shared characteristics between the corresponding DNA structures and slipped DNAs. Such features of the repeats may be recognized by cellular proteins known to bind such structures.
Tam
Mandy
M
Program of Genetics and Genomic Biology, The Hospital for Sick Children, Room 11-135, 555 University Avenue, Elm Wing, M5G 1X8, Toronto, Ont., Canada
Erin Montgomery
S
S
Kekis
Mariana
M
Stollar
B David
BD
Price
Gerald B
GB
Pearson
Christopher E
CE
D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000918:Antibody Specificity; D000067696:Ataxin-1; D000067528:Ataxins; D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009223:Myotonic Dystrophy / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D009705:Nucleosides / Q000276:immunology; D013499:Surface Properties; D018911:Trinucleotide Repeats / Q000276:immunology*
C000593749:ATXN1 protein, human; D000974:Antibodies, Antinuclear; D000067696:Ataxin-1; D000067528:Ataxins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D009705:Nucleosides; D004247:DNA; D008274:Magnesium