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. 2019 Jan;14(1):248-282.
doi: 10.1038/s41596-018-0093-7.

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

Jinchuan Hu  1   2 , Wentao Li  3 , Ogun Adebali  4 , Yanyan Yang  2 , Onur Oztas  2 , Christopher P Selby  2 , Aziz Sancar  5
Affiliations

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

Jinchuan Hu et al. Nat Protoc. 2019 Jan.

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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Figures

Fig. 1 |
Fig. 1 |. Schematic overview of the XR-seq protocol.
The red star denotes the DNA damage in the excised oligomer released during nucleotide excision repair. The purple and orange lines represent the 5′ and 3′ adapters, respectively. BPDE, benzo[a]pyrene diol epoxide; CDDP, cisplatin; NaCN, sodium cyanide; UV, ultraviolet.
Fig. 2 |
Fig. 2 |. Representative gels showing the excision assay and isolation of PCR products during XR-seq library preparation.
a, Image showing the excised oligomers containing (6–4)PPs at different time points after 10 J/m2 UV irradiation of A375 cells. For each lane, the excised oligomers were isolated by Hirt extraction from one 150-mm tissue culture dish of cells and then immunoprecipitated with anti-(6–4)PP antibody. Purified excised oligomers were 3′-end-labeled and resolved on a sequencing gel. Adapted from Hu et al. under a Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). b, Image showing the pilot PCR products from Step 29. Lanes 3, 4 and 5 show the pilot PCR products after damage reversal. The PCR products in Lane 4, as shown by the band intensity, represent the optimal number of PCR cycles, which are sufficient for NGS. On the basis of this image, the optimal number of PCR cycles for the library preparation is 12 (18 – 6 = 12). c,d, Images showing a gel used to isolate the final PCR products of two biological replicates before (c, Step 35) and after (d, Step 37) excision from the gel. The black asterisk denotes the PCR products of adapter dimers. e, Image showing dsDNA libraries for tXR-seq in a pilot PCR reaction. CPD and BPDE-dG damage were bypassed by DNA polymerases η and κ, respectively. Lanes 3 and 8 represent the optimum pilot PCR results, and based on this result, 12 (18 – 6 = 12) and 15 (21 – 6 = 15) PCR cycles were chosen for CPD and BPDE-dG tXR-seq, respectively. f, Length distribution of excised oligomers from CPD tXR-seq and CPD XR-seq. e,f adapted from Li et al., Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene. Proc. Natl. Acad. Sci. USA 114, 6752–6757 (2017). M, DNA size marker; NC, non-template control.
Fig. 3 |
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.
a, The read length distribution shows a single population (no degradation products) following TFIIH immunoprecipitation. b, Dithymine frequency is enriched at two main positions, 19–20 and 20–21, for the 26-nt excised oligomers obtained from CPD XR-seq. c, Screenshot of XR-seq signals in two genes, ZBTB4 and POLR2A, transcribed in opposite directions. Arrows indicate the direction of transcription. Repair asymmetry between strands that is due to transcription-coupled repair in the expressed genes is observed. The transcribed strands are the plus (above) and minus (below) strands for ZBTB4 and POLR2A, respectively. Therefore, those regions are repaired at higher levels because of transcription-coupled repair. The magnitude of the asymmetry varies depending on organism, gene and damage type. Data in c are from Hu et al.. Histograms were plotted using the R software and the ggplot2 package, and the genomic region-specific repair levels were captured using IGV.
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