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短识别序列 DNA 胞嘧啶 5-甲基转移酶的鉴定、表达与纯化。

Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences.

机构信息

Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.

Division of Infection and Immunity, Research Center for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan.

出版信息

BMC Biotechnol. 2022 Nov 4;22(1):33. doi: 10.1186/s12896-022-00765-3.

DOI:10.1186/s12896-022-00765-3
PMID:36333700
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9636781/
Abstract

BACKGROUND

DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences.

RESULTS

We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising.

CONCLUSIONS

In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

摘要

背景

DNA 甲基转移酶（MTases）是一种能诱导甲基化的酶，是 DNA 代表性的表观遗传修饰之一，也是分析表观基因组的有用工具。然而，就 DNA 胞嘧啶 5-甲基化而言，迄今为止鉴定的 MTases 存在一些缺点，即它们的识别序列与哺乳动物细胞中内源性 DNA 甲基化的识别序列重叠，或者识别序列太长，不适合精细的表观遗传图谱绘制。为了鉴定与 CG 二核苷酸永不重叠的短识别序列的 MTases，我们系统地研究了使用数据库搜索鉴定的 25 种候选酶，这些酶与已知识别短序列的胞嘧啶 5-MTases 具有高度相似性。

结果

我们鉴定了 6 种具有新识别序列的 MTases，包括 TCTG、CC、CNG、TCG、GCY 和 GGCA。由于识别序列永不与 CG 二核苷酸重叠，因此识别 CC 二核苷酸的 MTases 很有前途。

结论

在本研究中，我们建立了一种产生活性 CC 甲基化 MTases 的方法，并将其应用于核小体占有率和甲基组测序，以证明该酶在精细的表观遗传图谱绘制中的有用性。永不与 CG 二核苷酸重叠的 MTases 将使我们能够同时分析多个表观基因组。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/ae9a4a518eb3/12896_2022_765_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/c66616e969b0/12896_2022_765_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/2b4a5d99d9e1/12896_2022_765_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/ab8d4309ab73/12896_2022_765_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/87f16d6853b4/12896_2022_765_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/23145e88619c/12896_2022_765_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/ae9a4a518eb3/12896_2022_765_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/c66616e969b0/12896_2022_765_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/2b4a5d99d9e1/12896_2022_765_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/ab8d4309ab73/12896_2022_765_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/87f16d6853b4/12896_2022_765_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/23145e88619c/12896_2022_765_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cfa8/9636781/ae9a4a518eb3/12896_2022_765_Fig6_HTML.jpg

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