title: Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA-DNA heteroduplex substrates.
abstract: The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA-RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA-DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA-DNA heteroduplex context and whether a RNA-DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA-DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA-DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.
authors.affiliation: CSIRO Food and Nutritional Sciences, North Ryde, Sydney, NSW 2113, Australia. jason.ross@csiro.au
authors.forename: Jason P
authors.initials: JP
authors.lastname: Ross
authors.forename: Isao
authors.initials: I
authors.lastname: Suetake
authors.forename: Shoji
authors.initials: S
authors.lastname: Tajima
authors.forename: Peter L
authors.initials: PL
authors.lastname: Molloy
mesh_terms: D018899:CpG Islands; D019175:DNA Methylation; D020868:Gene Silencing; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism
chemical_list: D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D013431:Sulfates; C012036:sodium sulfate; D008780:Methyltransferases