Evaluation of the Properties of the DNA Methyltransferase from Aeropyrum pernix K1.
Evaluation of the Properties of the DNA Methyltransferase from Aeropyrum pernix K1.
复制标题
Aeropyrum pernix K1 DNA甲基转移酶的性质评价
DOI：
10.1128/spectrum.00186-21
复制
发表时间：
2021-10-31
影响因子：
3.7
通讯作者：
Iida Y
中科院分区：
文献类型：
--
作者：
Hayashi M;Sugahara K;Yamamura A;Iida Y
关于超嗜热古菌的DNA甲基转移酶（MTases），我们所知甚少。在本研究中，我们重点研究了一种来自于热液喷口的超嗜热古菌Aeropyrum pernix K1的MTase，其最佳生长温度为90°C ~ 95°C。通过基因组序列分析，推测冬姜K1具有限制性修饰系统（R-M系统）。来自A. pernix K1的限制性内切酶（New England BioLabs Inc.[目录代码R06435]称为ApeKI）先前已被描述，但来自A. pernix K1 （m.a ApeKI）的MTase的性质尚未澄清。因此，我们证明了m.a apeki的性质。在本研究中，M.ApeKI在大肠杆菌JM109中表达，并利用其His标签进行亲和纯化。通过甲基化活性和亚硫酸盐测序（BS-seq）确定m.a apeki的识别序列。采用高效液相色谱法（HPLC）检测甲基化胞嘧啶的位置。结果表明，M.ApeKI在5 ‘ -GCWGC-3 ’中第二个胞嘧啶的C-5位置添加了甲基。此外，我们还确定了MTase的最适温度在70°C以上，并且它对高温有很强的耐受性。m.a apeki是第一个高度耐热的DNA（胞嘧啶-5）甲基转移酶，通过实验证据进行评估。一般来说，在最佳生长温度超过或等于60°C的嗜热细菌中，预测其DNA中的甲基化碱基仅包括n4 -甲基胞嘧啶或n6 -甲基腺嘌呤，因为5-甲基胞嘧啶容易被热脱胺。然而，从本研究中，在95°C的最佳生长温度下，证明了a . pernix K1产生DNA（胞嘧啶-5）甲基转移酶。因此，A. pernix K1可能在其DNA中含有5-甲基胞嘧啶，并可能为预期的C-to-T突变产生原始修复系统。m.a apeki已被证明耐高温；因此，我们期望m.a apeki可能对开发新的分析系统或表观遗传编辑工具有价值。
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 N4-methylcytosine or N6-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.