Abstract
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.
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Acknowledgements
We thank A. Kakoki for critical reading of the manuscript. This work was supported by National Institutes of Health grants GM118102 and ES027255 (to A.S.) and Scientific and Technological Research Council of Turkey grant 118C023 (to O.A.).
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J.H., W.L., O.A., Y.Y., O.O. and C.P.S. performed the experiments and analyzed the data described in the protocol. W.L. wrote the manuscript with assistance from C.P.S, J.H., O.A., Y.Y., O.O. and A.S. All authors contributed to, reviewed and approved the manuscript.
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Key references using this protocol
Hu, J., Adar, S., Selby, C. P., Lieb, J. D. & Sancar, A. Genes Dev. 29, 948–960 (2015): http://www.genesdev.org/cgi/doi/10.1101/gad.261271.115
Li, W. et al. Proc. Natl. Acad. Sci. USA 114, 6752–6757 (2017): https://doi.org/10.1073/pnas.1706021114
Adebali, O., Chiou, Y.-Y., Hu, J., Sancar, A. & Selby, C. P. Proc. Natl. Acad. Sci. USA 114, E2116–E2125 (2017): https://doi.org/10.1073/pnas.1700230114
Yang, Y. et al. Proc. Natl. Acad. Sci. USA 115, E4777–E4785 (2018): https://doi.org/10.1073/pnas.1804493115
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Hu, J., Li, W., Adebali, O. et al. Genome-wide mapping of nucleotide excision repair with XR-seq. Nat Protoc 14, 248–282 (2019). https://doi.org/10.1038/s41596-018-0093-7
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DOI: https://doi.org/10.1038/s41596-018-0093-7
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