| Autor: |
Hayashi M; Department of Applied Chemistry and Bioscience, Graduate School of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan., Sugahara K; Department of Applied Bioscience, Faculty of Applied Bioscience, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan., Yamamura A; Department of Applied Chemistry and Bioscience, Graduate School of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan.; Department of Applied Bioscience, Faculty of Applied Bioscience, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan., Iida Y; Department of Applied Chemistry and Bioscience, Graduate School of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan.; Department of Applied Bioscience, Faculty of Applied Bioscience, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan. |
| Abstrakt: |
Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N 4 -methylcytosine or N 6 -methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool. |
