Nucleotide excision repair in Human cell lines lacking both XPC and CSB proteins

Abstract

Nucleotide excision repair removes UV-induced DNA damage through two distinct sub-pathways, global repair and transcription-coupled repair (TCR). Numerous studies have shown that in human and other mammalian cell lines that the XPC protein is required for repair of DNA damage from nontranscribed DNA via global repair and the CSB protein is required for repair of lesions from transcribed DNA via TCR. Therefore, it is generally assumed that abrogating both sub-pathways with an XPC-/-/CSB-/- double mutant would eliminate all nucleotide excision repair. Here we describe the construction of three different XPC-/-/CSB-/- human cell lines that, contrary to expectations, perform TCR. The XPC and CSB genes were mutated in cell lines derived from Xeroderma Pigmentosum patients as well as from normal human fibroblasts and repair was analyzed at the whole genome level using the very sensitive XR-seq method. As predicted, XPC-/- cells exhibited only TCR and CSB-/- cells exhibited only global repair. However, the XPC-/-/CSB-/- double mutant cell lines, although having greatly reduced repair, exhibited TCR. Mutating the CSA gene to generate a triple mutant XPC-/-/CSB-/-/CSA-/- cell line eliminated all residual TCR activity. Together, these findings provide new insights into the mechanistic features of mammalian nucleotide excision repair.

Graphical Abstract

Sensitive whole-genome repair mapping reveals unexpected repair activity in XPC-/-/CSB-/- cells constructed to eliminate both sub pathways of nucleotide excision repair.

Figure 1.

Transcription-coupled repair in patient-derived CS-B/XPC^−/− cells. (A, B) CPD XR-seq data from the indicated cell lines is plotted as average repair reads (y-axis) along the length of a ‘unit gene’ (x-axis) (divided into 100 bins; 10100 genes were selected for length >5 kb and no overlaps with a distance of at least 5 kb between genes). (A) As predicted for repair of CPDs in humans, normal human fibroblasts (NHF1) exhibit only a small amount of TCR due to predominant global repair, patient-derived XP-C cells which lack the XPC protein required for global repair exhibit only TCR, and patient-derived CS-B cells which lack the CSB protein required for TCR exhibit only global repair. (B) Unexpectedly, when XPC is mutated in the patient-derived CS-B cells, transcription-coupled repair is now detectable. RPKM, reads per kilobase per million mapped reads; TSS, transcription start site; TES, transcription end site. TS, transcribed strand; NTS, nontranscribed strand. (C) Repair as a function of genomic location. The parental patient-derived CS-B cell line, which exhibits only global repair, has the largest proportion of repair events in intergenic regions, whereas in the CS-B/XPC^−/− cells most of the repair reads map to genes, consistent with repair by TCR.

Figure 2.

Transcription-coupled repair in normal human fibroblast double knockout NHF1/XPC^−/−/CSB^−/− cells. (A) CPD XR-seq data from the indicated NHF1 cell lines is plotted as in Figures1A, B and shows a similar CPD repair pattern in normal human fibroblasts as was seen in the patient-derived cells. (B) CPD repair as a function of genomic location in NHF1 cells was analyzed as in Figure 1C.

Figure 3.

NHF1/XPC^−/−/CSB^−/− cells excise UV photoproducts by the same dual incision mechanism as wild type cells. (A) Length distribution of XR-seq reads from the indicated NHF1 cell lines mapped to either nuclear DNA (left) or mitochondrial DNA (right). The 26-nt median is indicated with a dashed line. (B) Analysis of the frequency of each of possible dipyrimidine along XR-seq reads of 26-nt length mapped as in (A). Mitochondrial DNA analysis and unirradiated cells (WT no UV, performed on the same 100× scale as the double knockout cells) were included to control for specificity.

Figure 4.

Extreme UV sensitivity and nearly undetectable repair in the XPC^−/−/CSB^−/− cell lines (A) The four NHF1 cell lines were analyzed for survival two days after the indicated doses of UVC. Shown are the mean from three biological replicates with error bars denoting standard deviation (SD). (B) Slot blot analysis showing CPD repair rates of the four NHF1 cell lines treated with 5 J/m² UVC. CPD signals were normalized to time = 0 and plotted as a function of time. Results shown are the mean from three biological replicates with error bars denoting SD. (C) The in vivo excision assay was used to compare the amount of CPD-containing excised oligos in extracts from three the parental and XPC^−/−/CSB^−/− cell lines (double knockouts in lanes 5, 7, 9 with the parental strain indicated above). An equal number of cells were irradiated with 20 J/m² UVC and incubated 2 h at 37°C to allow for repair. Cells were lysed by the Hirt procedure and low molecular weight DNA in the supernatant was immunoprecipitated with anti-CPD antibodies. The recovered oligos were mixed with a 50-mer internal control oligo, 3′-end labeled, and separated on a DNA sequencing gel along with the indicated size markers. (D) Quantification of three biological replicates of the excision assays above showing the mean with error bars denoting SD.

Figure 5.

Mutating the CSA gene to generate a triple mutant NHF1/XPC^−/−/CSB^−/−/CSA^−/− cell line eliminates TCR activity. qXR-seq data plotted as in Figures 1 and 2 show that UV-induced (6–4)PP damage is repaired by TCR in XPC^−/−/CSB^−/− cells (middle), but not in WT NHF1 (left) or XPC^−/−/CSB^−/−/CSA^−/− cells (right).

Figure 6.

Model for global and transcription-coupled repair (TCR) of UV-induced DNA damage in human cells lacking XPC and CSB proteins. In global repair (left), in the absence of XPC, which is represented as a gray dashed oval, the dual incision complex is assembled at a damage site by binding of XPA-RPA to damage and recruitment of TFIIH by its interaction with XPA. Recruitment of the XPG and XPF nucleases enables dual incisions generating a 26–27-nt long oligomer. In TCR (right), in the absence of CSB, represented as a gray dashed oval, the transcription bubble replaces the XPC damage recognition-function and enables the assembly of the 5 excision factors (XPA, RPA, TFIIH, XPG, and XPF). Excision occurs in the absence of XPC and CSB, but it is drastically reduced because assembly of the 5-factor nuclease is inefficient without these two proteins. Adapted with permission from (43).