해양 환경에서 분리한 Celeribacter marinus IMCC12053의 핵산염기 외향고리 메틸화 효소 연구

Abstract

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, has N6- adenine or N4-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 and Novosphingobium pentaromativorans US6-1 isolated from the marine environment are alphaproteobacteria. Both strains replace the methyl groups of the exocyclic amines of CpG and GpC cytosines to produce N4-methyl cytosine. Using single molecule real-time sequencing method (SMRT), methylation patterns of C. marinus IMCC12053 and N. pentaromativorans US6-1 were compared using Gibbs motif sampler program. Both strains showed conversion of adenosine of 5’-GANTC-3’ to N6-methyladenosine, and N4-cytosine of 5’-CpG-3’ (IMCC12053) and 5’-GpC-3’ (US6-1) to N4-methylcytosine. Exocylic DNA methyltransferases from both of the species were chosen for cloning using phylogenetic analysis. IPTG induction were performed to confirm the methylation activity of the ORF’s from the two strains when they were cloned into pQE30 vector. The genomic DNA and plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes sensitive to methylation to confirm the methylation activity. These methylases protected the restriction enzyme site by IPTG induced changes in the DNA. Thus the recognition sequences cleaved by restriction enzymes are decreased and DNA fragments protected by methylation increased in accordance with the cytosine methylation. In this study, characteristics of cloned exocyclic DNA methylases are investigated for potential uses of novel type of CpG methylase for molecular biology and epigenetics.