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Genome-wide mapping of nucleotide excision repair with XR-seq

Nature Protocols volume 14, pages 248–282 (2019)Cite this article

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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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Fig. 1: Schematic overview of the XR-seq protocol.
Fig. 2: Representative gels showing the excision assay and isolation of PCR products during XR-seq library preparation.
Fig. 3: Preliminary analyses of CPD XR-seq data at a 1-h time point from treatment of human NHF1 cells with 10 J/m2 UV.

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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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  1. These authors contributed equally: Jinchuan Hu, Wentao Li.

Authors and Affiliations

  1. The Fifth People’s Hospital of Shanghai and Institute of Biomedical Sciences, Fudan University, Shanghai, China

    Jinchuan Hu

  2. Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Jinchuan Hu, Wentao Li, Yanyan Yang, Onur Oztas, Christopher P. Selby & Aziz Sancar

  3. Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey

    Ogun Adebali

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Contributions

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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Correspondence to Wentao Li or Aziz Sancar.

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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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