Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India; Department of Zoology, Centre for Advanced Studies, Savitribai Phule Pune University, Pune 411007, India.
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India.
Biochim Biophys Acta Gen Subj. 2017 Mar;1861(3):593-602. doi: 10.1016/j.bbagen.2016.12.025. Epub 2016 Dec 27.
Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans.
C-5 methyl cytosines (mC) were detected by immuno-blotting with mC-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry.
Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles mC methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost mC modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc.
DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing mC.
Although, dispensable under optimal growth conditions, the presence of mC may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.
在生物体中，通过 DNA 中胞嘧啶的表观遗传修饰来控制细胞过程是广泛存在的，但在极其耐辐射的微生物 D. radiodurans 中，这种现象迄今尚不清楚。
用 mC 特异性抗体进行免疫印迹检测 C-5 甲基胞嘧啶（mC）。通过亚硫酸氢盐测序研究由 DR_C0020 编码的蛋白质对胞嘧啶甲基化的位点。通过定点突变构建了 DR_C0020 敲除突变体（Δdcm），并评估其对生长、辐射抗性和蛋白质组的影响。蛋白质通过质谱鉴定。
在 D. radiodurans 基因组中检测到甲基化的胞嘧啶。DR_C0020 编码的蛋白（Dcm，NCBI 登录号：WP_034351354.1），其氨基酸序列类似于 mC 甲基转移酶，被证明是唯一的 SAM 依赖性 C-5 胞嘧啶甲基转移酶。纯化的 Dcm 蛋白被发现甲基化 CpN 序列，并且优先甲基化两个连续的胞嘧啶。Δdcm 菌株完全失去了基因组中的 mC 修饰，对生长没有影响，但变得对辐射敏感。Δdcm 细胞中涉及蛋白质稳态、氧化应激防御、代谢等的几种蛋白质的丰度发生了微小变化。
DR_C0020 编码的 SAM 依赖性甲基转移酶 Dcm 是负责 D. radiodurans 基因组中 CpN 位点 C-5 胞嘧啶甲基化的唯一酶，并且在正常生长条件下调节蛋白质稳态。该蛋白质是一种不寻常的氨基酸甲基转移酶，已经进化为产生 mC。
尽管在最佳生长条件下是可有可无的，但 mC 的存在可能对识别亲本链很重要，因此可能有助于 D. radiodurans 中异常的 DNA 修复。
