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用于胸腺嘧啶乙二醇全基因组定位的DNA蛋白质交联测序
作者:小柯机器人 发布时间:2022/1/5 15:15:47

武汉大学袁必锋团队开发了用于胸腺嘧啶乙二醇全基因组定位的DNA蛋白质交联测序方法。相关研究成果发表在2022年1月3日出版的《美国化学会杂志》。

胸腺嘧啶乙二醇(Tg)是DNA中最常见的氧化诱导嘧啶损伤形式。Tg可由DNA中胸苷的直接氧化引起。此外,5-甲基-2′-脱氧胞苷(5-mdC)可被氧化为5-mdC乙二醇,其随后的脱氨作用也产生Tg。然而,Tg在人类基因组中的分布仍然未知。

该文中,研究人员提出了一种DNA-蛋白质交联测序(DPC-Seq)方法,用于人类细胞中Tg的全基因组定位。该方法利用双功能DNA糖基化酶(即NTHL1)的特异性进行共价标记,以及DPC下拉、SDS-PAGE分离和膜转移,高效和选择性地富集含Tg的DNA。通过使用DPC-Seq,在人类基因组中检测到数千个Tg位点,其中NTHL1和NEIL1(负责Tg修复的主要DNA糖化酶)的双重消融导致Tg峰的数量显著增加。此外,Tg在与活跃转录相关的基因组区域缺失,但在核小体结合位点富集,特别是在以H3K9me2标记的异染色质位点。研究人员共同开发了一种DPC-Seq方法,用于高效富集含Tg的DNA,并用于在人类细胞中绘制Tg的全基因组图谱。

研究工作为今后DNA中Tg的功能研究提供了一个强有力的工具,并且研究人员认为该方法也可以在将来用于基因组DNA中其他修饰核苷图谱的绘制。

附:英文原文

Title: DNA–Protein Cross-Linking Sequencing for Genome-Wide Mapping of Thymidine Glycol

Author: Feng Tang, Jun Yuan, Bi-Feng Yuan, Yinsheng Wang

Issue&Volume: January 3, 2022

Abstract: Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesions in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2′-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol, and its subsequent deamination also yields Tg. However, Tg's distribution in the human genome remains unknown. Here, we presented a DNA–protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach capitalizes on the specificity of a bifunctional DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in the human genome, where dual ablation of NTHL1 and NEIL1, the major DNA glycosylases responsible for Tg repair, led to pronounced increases in the number of Tg peaks. In addition, Tg is depleted in genomic regions associated with active transcription but enriched at nucleosome-binding sites, especially at heterochromatin sites marked with H3K9me2. Collectively, we developed a DPC-Seq method for highly efficient enrichment of Tg-containing DNA and for genome-wide mapping of Tg in human cells. Our work offers a robust tool for future functional studies of Tg in DNA, and we envision that the method can also be adapted for mapping other modified nucleosides in genomic DNA in the future.

DOI: 10.1021/jacs.1c10490

Source: https://pubs.acs.org/doi/10.1021/jacs.1c10490

 

期刊信息

JACS:《美国化学会志》,创刊于1879年。隶属于美国化学会,最新IF:14.612
官方网址:https://pubs.acs.org/journal/jacsat
投稿链接:https://acsparagonplus.acs.org/psweb/loginForm?code=1000