检测基因组上氧化损伤准确位置的高通量测序方法的开发和应用

Both exogenous and endogenous oxidative stress can induce oxidative DNA damage such as 8-oxo-dG which can cause mutation and result in cancer and/or aging. Damage and mutation located on different positions of genome may have quite different biological effect, however, very little is known about this topic. Although a couple of methods for mapping oxidative damage were reported in recent years, they have limited practicability due to various weakness of each method. The applicant has developed a high-throughput sequencing-based method named Damage-seq to map the precise positions of bulky adducts in the whole human genome. Based on this method, this study will label 8-oxo-dG with biotin by a chemical reaction, which enables the enrichment of labeled DNA by streptavidin-coated magnetic beads and the blockage of DNA polymerase by the labeled nucleotides. These two features will make it possible to use a modified Damage-seq procedure to detect biotin-labeled 8-oxo-dG. This new method will be used to study how chromatin environment such as spatial distribution, accessibility and protein binding influences the formation of oxidative damage and how transcription affects the repair of oxidative damage. This project aims to develop a new method to precisely detect the distribution of oxidative damage in the human genome, which will provide a new tool as well as a novel perspective for the study of oxidative damage and its repair.

外源或者内源的氧化压力会产生以8-oxo-dG为代表的DNA氧化损伤，它会导致DNA突变，并且与癌症和衰老的发生有密切的关系。发生在基因组上不同位置的损伤和突变所引起的后果也是截然不同的，但目前对这方面所知甚少。尽管近年来已经报道了多种检测氧化损伤的测序方法，但它们都存在一些缺陷，实用价值有限。申请人在前期工作中建立了Damage-seq方法，可以检测基因组上DNA加合物损伤的准确位置。在此基础上，本研究将通过化学反应在8-oxo-dG上标记生物素，使其可以被链霉素磁珠富集并阻挡DNA聚合酶的延伸，从而可以用Damage-seq来检测。本项目将利用这种新方法，研究染色质环境如DNA在核内的空间分布、可接近性和蛋白结合等因素对氧化损伤形成的影响，以及转录对氧化损伤修复的影响。本项目的实施，预期将能够准确检测人类基因组上氧化损伤的分布，为氧化损伤修复的研究提供新的工具和视角。

8-Oxo-7,8-dihydro-2’-deoxyguanosine（OG）是最常见的DNA氧化损伤之一，其可导致基因组不稳定，并与癌症，神经系统疾病和衰老有关。OG及其修复中间体也可以调节基因转录，从而在细胞氧化应激过程发挥作用。然而，目前还缺乏能精确和特异地检测基因组上OG分布的技术，阻碍了对OG的生物学功能的研究。在本项目中，我们开发了一种单碱基分辨率OG测序方法CLAPS-seq（Chemical Labeling And Polymerase Stalling Sequencing）。该方法利用一个选择性化学反应给OG标记上生物素并用链霉亲和素磁珠捕捉和纯化带有OG的DNA片段，随后根据生物素标记的OG能阻挡DNA聚合酶的性质，用引物延伸反应来定位OG的准确位置。CLAPS-seq可以准确测量HeLa细胞外源性和内源性OG的全基因组分布，各条件下在检测到的损伤位点上G的比例都达到或超过70%，证明该方法具有很高的特异性。从测序数据中我们发现G-四链体（G4）上OG发生率显著下降，可能是导致具有高GC含量的转录起始位点附近区域OG反而减少的原因之一。此外，我们发现潜在的G-四链体序列（PQS）是OG的热点。由于OG可以促进PQS转换为G4，这提示未形成G4结构的PQS（non-G4 PQSs）可能通过转换为G4来促进下游基因表达，从而在OG介导的氧化应激反应中起一定作用。.此外，我们将CLAPS-seq的策略应用到基因组上尿嘧啶的检测，开发了单碱基分辨率尿嘧啶测序技术Ucaps-seq，并利用该方法找到了一个以前从未报道过的胞嘧啶碱基编辑器BE4max在编辑RNF2位点时的脱靶位点，表明Ucaps-seq在评估胞嘧啶碱基编辑器的特异性方面具有潜在的应用价值。