A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA

• Main Authors: Gao, M. (Author), Hu, J. (Author), Huang, Y. (Author), Sancar, A. (Author), Selby, C.P (Author), Wu, S. (Author)
• Format: Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology Inc. 2022
• Subjects: Antibodies; Complex procedure; Dimer formation; Dimers; DNA; Excision repair; Genes; Immunoprecipitations; Immunopurification; Mapping; New approaches; Nucleotide excision repair; Nucleotides; Polymerase chain reaction; Repair functions; Simple++; Single nucleotides
• Online Access: View Fulltext in Publisher

 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)–XRseq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 30-dA-tailing and 50-adapter ligation instead of the original 50- and 30-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. © 2022 THE AUTHORS.