Nucleotide excision repair of abasic DNA lesions

Abstract

Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.

Figure 1.

Impairment of transcription by BER-resistant AP lesion positioned at a specific nucleotide in the transcribed strand of the EGFP gene. (A) Structures of synthetic tetrahydrofuran (THF and S-THF) AP lesions and reactivity of BER enzymes towards the specified types of AP sites. (B) Characterization of reporter constructs containing deoxyguanine (dG) or the specified types of AP lesion at a defined nucleotide (*) in the transcribed DNA strand (TS). Scheme shows position for incorporation of synthetic oligonucleotides containing dG, THF or S-THF with respect to EGFP coding sequence (arrow) and transcription start (broken arrow). To demonstrate the presence of AP lesion, the obtained constructs were incubated with excess of APE1 and analysed by gel electrophoresis in the presence of ethidium bromide. See also Supplementary Figure S1 for more detail. (C) Flow cytometry analyses of expression of constructs containing specified modifications in transfected XP-A (GM04312) cells (a representative experiment). EGFP fluorescence distribution plots show expression data overlaid pairwise for each modification and the respective control constructs without modification. Bar chart on the right shows quantification of the EGFP expression, relative to the matched control constructs without the modifications.

Figure 2.

Reactivation of expression constructs containing AP lesions by complementation with XPA. (A) Flow cytometry expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF(N²)-dG (rose) at the analysed position in transcribed DNA strand of the EGFP gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF(N²)-dG samples overlaid with a common dG reference sample. (B) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P-values calculated by the Student’s t-test). See also Supplementary Figure S2.

Figure 3.

Impact of defects in different NER genes on the removal of the transcription-blocking S-THF lesion. (A) HCR of the expression constructs containing S-THF or the AAF(N²)-dG adduct in the XPF KO and the isogenic MRC-5 Vi cells. EGFP versus DsRed scatter plots and the derived EGFP fluorescence distribution plots (overlaid with the respective control ‘dG’ constructs). (B) HCR of the expression construct containing S-THF in human skin fibroblast cell lines of the specified NER complementation groups. Overlaid fluorescent distribution plots were generated as described above but scatter plots were omitted for clarity of presentation. (C) Quantification of expression of constructs containing the S-THF AP lesion relative to the dG reference (mean of n independent experiments ± SD; P-values calculated by the Student’s t-test). The MRC-5 value shows pulled data for two independent clones (see ‘Material and Methods’ section).

Figure 4.

Transcriptional mutagenesis at the BER-resistant abasic site in the template DNA and its suppression by NER. (A) Scheme of the reporter for detection of ribonucleotide misincorporation opposite to AP-lesion in the template DNA. Substitution of 613U in mRNA to any other ribonucleotide results in reversion to a fluorescent EGFP. (B) Flow cytometry assay for detection of the mRNA single nucleotide substitutions induced by the specified AP lesions (THF, S-THF) in the MRC-5 (group of panels on the left) and XP-A (group of panels on the right) cell lines. Fluorescence scatter plots show full data for individual samples from a representative experiment. The derived EGFP fluorescence distribution plots show overlaid data for EGFP construct without modification (green colour) and EGFP Q205* constructs without modification (blue) or with the indicated lesion (amber). The nature of the nucleotide/modification in the template DNA strand is indicated above the plots. Note the right shift of S-THF plots compared to dA.

Figure 5.

Transcriptional mutagenesis by THF and S-THF lesions in the panel of NER deficient cell lines: overlaid fluorescence distribution plots from a representative experiment and a bar chart showing quantification of the EGFP expression of the specified pEGFP Q205* constructs, relative to the original EGFP without modification in the transcribed DNA strand (ts.613G). Data of four independent experiments (mean ±SD; P-values calculated by the Student's t-test).