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KMID : 0364820190550020103
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Korean Journal of Microbiology
2019 Volume.55 No. 2 p.103 ~ p.111
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Exocyclic GpC DNA methyltransferase from Celeribacter marinus IMCC12053
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Kim Jung-Hee
Oh Hyun-Myung
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Abstract
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DNA methylation is involved in diverse processes in bacteria,including maintenance of genome integrity and regulation ofgene expression. CcrM, the DNA methyltransferase conservedin Alphaproteobacterial species, carries out N6-adenine or N4-cytosine methyltransferase activities using S-adenosyl methionineas a co-substrate.
Celeribacter marinus IMCC12053 from the Alphaproteobacterialgroup was isolated from a marine environment. Single moleculereal-time sequencing method (SMRT) was used to detect themethylation patterns of C. marinus IMCC12053. Gibbs motifsampler program was used to observe the conversion of adenosineof 5'-GANTC-3' to N6-methyladenosine and conversion of N4-cytosine of 5'-GpC-3' to N4-methylcytosine. Exocyclic DNAmethyltransferase from the genome of strain IMCC12053 waschosen using phylogenetic analysis and N4-cytosine methyltransferasewas cloned. IPTG inducer was used to confirm themethylation activity of DNA methylase, and cloned into apQE30 vector using dam-/dcm- E. coli as the expression host.
The genomic DNA and the plasmid carrying methylase-encodingsequences were extracted and cleaved with restriction enzymesthat were sensitive to methylation, to confirm the methylationactivity. These methylases protected the restriction enzyme siteonce IPTG-induced methylases methylated the chromosomeand plasmid, harboring the DNA methylase. In this study, clonedexocyclic DNA methylases were investigated for potential useas a novel type of GpC methylase for molecular biology andepigenetics.
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KEYWORD
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adenosine, cytosine, DNA methyltransferase, exocyclic amine group
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