The Fifth People's Hospital of Shanghai and Institute of Biomedical Sciences, Fudan University, Shanghai, China.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
Nat Protoc. 2019 Jan;14(1):248-282. doi: 10.1038/s41596-018-0093-7.
Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed excision repair sequencing (XR-seq) to study nucleotide excision repair of DNA adducts in humans, mice, Arabidopsis thaliana, yeast and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction, are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adapters and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a double-stranded DNA (dsDNA) form by using appropriate translesion DNA synthesis (TLS) polymerases and then can be amplified by PCR. The current genome-wide approaches for studying repair measure the loss of damage signal with time, which limits their resolution. By contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7-9 d.
核苷酸切除修复是一种修复多种大体积 DNA 加合物的多功能机制。我们开发了切除修复测序（XR-seq）技术，以研究人类、小鼠、拟南芥、酵母和大肠杆菌中 DNA 加合物的核苷酸切除修复。在本方案中，通过细胞裂解和分级分离分离出核苷酸切除修复反应中产生的切除寡聚物，然后用损伤或修复因子特异性抗体从非染色质部分进行免疫沉淀。单链切除寡聚物与接头连接，并使用损伤特异性抗体再次进行免疫沉淀。然后通过酶或化学反应逆转切除寡聚物中的 DNA 损伤，然后通过 PCR 扩增将其转化为测序文库。或者，含有 DNA 损伤的切除寡聚物，特别是含有不可逆 DNA 损伤的寡聚物，如苯并[a]芘诱导的 DNA 加合物，可以通过使用适当的跨损伤 DNA 合成（TLS）聚合酶转化为双链 DNA（dsDNA）形式，然后通过 PCR 进行扩增。目前用于研究修复的全基因组方法通过时间来衡量损伤信号的损失，这限制了它们的分辨率。相比之下，XR-seq 的一个优势是修复信号可以直接在零背景上检测到。使用这里描述的方案获得的 XR-seq 文库可以在 7-9 天内获得。
