A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA.
A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA.
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一种无需对受损 DNA 进行免疫纯化的全基因组核苷酸切除修复作图新技术
DOI：
10.1016/j.jbc.2022.101863
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Hu, Jinchuan
中科院分区：
文献类型：
--
作者：
Wu, Sizhong;Huang, Yanchao;Selby, Christopher P.;Gao, Meng;Sancar, Aziz;Hu, Jinchuan
核苷酸切除修复功能可以保护基因组的完整性，正在进行的使用切除修复测序（XR-seq）的研究有助于我们理解细胞如何优先修复整个基因组。在这种方法中，切除修复的产物承载受损的DNA被捕获，测序，然后以单核苷酸分辨率在全基因组范围内作图。然而，试剂要求和复杂的程序限制了该技术的广泛使用。除了这些试剂的费用之外，已经假设使用针对受损DNA的抗体的免疫沉淀步骤可能在不同的序列环境中引入偏倚。在这里，我们描述了一种新开发的适应称为dA-tailing and adaptor ligation（ATL）-XR-seq，这是一种相对简单的XR-seq方法，避免了使用针对受损DNA的免疫沉淀。ATL-XR-seq通过3′-dA加尾和5′-接头连接而不是原始的5′-和3′-双接头连接来捕获修复产物。这种新方法避免了在随后的PCR过程中形成衔接子二聚体，省略了低效和耗时的纯化步骤，并且非常灵敏。此外，poly（dA）尾长异质性可以作为分子标识符，允许映射更多的修复热点。重要的是，两种修复作图方法的比较表明，原始XR-seq程序中使用的抗UV损伤抗体没有引入重大偏倚。最后，我们还将所描述的DA加尾方法与定量PCR结合在一种新的方法中来定量修复产物。这些新的方法提供了强大的和用户友好的工具，定性和定量测量切除修复。
Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)–XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3′-dA-tailing and 5′-adapter ligation instead of the original 5′- and 3′-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.
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影响因子：
64.8
作者：
Ernst, Jason;Kheradpour, Pouya;Mikkelsen, Tarjei S.;Shoresh, Noam;Ward, Lucas D.;Epstein, Charles B.;Zhang, Xiaolan;Wang, Li;Issner, Robbyn;Coyne, Michael;Ku, Manching;Durham, Timothy;Kellis, Manolis;Bernstein, Bradley E.
通讯作者：
Bernstein, Bradley E.
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
14.9
作者：
Choi JH;Gaddameedhi S;Kim SY;Hu J;Kemp MG;Sancar A
通讯作者：
Sancar A
影响因子：
13.6
作者：
Jin SG;Pettinga D;Johnson J;Li P;Pfeifer GP
通讯作者：
Pfeifer GP
DOI：
10.1016/j.jbc.2021.100581
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jiang Y;Li W;Lindsey-Boltz LA;Yang Y;Li Y;Sancar A
通讯作者：
Sancar A