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Nucleic Acids Res. 2015 Nov 16;43(20):10055-64. doi: 10.1093/nar/gkv1008. Epub 2015 Oct 1.

Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation.

Author information

1
Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
2
Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada.
3
UW Medicine South Lake Union, University of Washington, Seattle WA 98109, USA.
4
Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada cepearson.sickkids@gmail.com.

Abstract

Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K(+), Na(+) or Li(+)), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.

PMID:
26432832
PMCID:
PMC4787773
DOI:
10.1093/nar/gkv1008
[Indexed for MEDLINE]
Free PMC Article

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