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Genome-wide systematic identification of methyltransferase recognition and modification patterns.

ABSTRACT: Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

SUBMITTER: Jensen TO

PROVIDER: S-EPMC6700114 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Jensen Torbjørn Ølshøj TØ Tellgren-Roth Christian C Redl Stephanie S Maury Jérôme J Jacobsen Simo Abdessamad Baallal SAB Pedersen Lasse Ebdrup LE Nielsen Alex Toftgaard AT

Nature communications 20190819 1

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potent ...[more]

PMID: 31427571

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Project description:BackgroundThe proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear.Methodology/principal findingsWe have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5') to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.Conclusion/significanceTaken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.

Dataset Information

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Publications

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

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