ERCC1-XPF targeting to psoralen-DNA crosslinks depends on XPA and FANCD2

Abstract

The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1-XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1-XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1-XPF recruitment to ICLs is less well understood. Moreover, specific mutations in XPF have been found to disrupt its function in ICLR but not in NER, but whether this involves differences in lesion targeting is unknown. Here, we imaged GFP-tagged ERCC1, XPF and ICLR-defective XPF mutants to investigate how in human cells ERCC1-XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. Our results confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2. Interestingly, we find that two ICLR-defective XPF mutants (R689S and S786F) are less well recruited to ICLs. These studies highlight the differential mechanisms that regulate ERCC1-XPF activity in DNA repair.

Keywords: DNA damage response; DNA repair; Fanconi anemia; Xeroderma pigmentosum–Cockayne syndrome complex.

Fig. 1

Generation of XPA knockout in ERCC1–GFP- and XPF–GFP-expressing cells. a Immunoblot showing XPA expression levels in XPA-proficient and XPA-knockout (XPA KO) U2OS cells expressing GFP-tagged ERCC1. SNF2H was used as loading control. b Immunoblot showing XPA expression levels in XPA-proficient and XPA-knockout (XPA KO) U2OS cells expressing GFP-tagged XPF. Tubulin was used as loading control. c Clonogenic UV survival assays of wild-type U2OS cells without any transgene (U2OS) and XPA-proficient and XPA KO U2OS cells expressing ERCC1–GFP. Results are plotted as average of three independent experiments, each performed in triplicate. d Clonogenic UV survival assays of XPA-proficient and XPA KO U2OS cells expressing XPF–GFP. Results are plotted as average of three independent experiments, each performed in triplicate. In c and d error bars represent the SEM

Fig. 2

XPF recruitment to LUD is XPA dependent. a Immunofluorescence images showing LUD recruitment of ERCC1–GFP (top panel) or XPF–GFP (lower panel) and endogenous XPA in XPA-proficient and XPA KO U2OS cells, 1 h after 60 J/m² UVC irradiation through an 8-µm microporous filter. Cells were immunostained against XPA and CPD, as damage marker. The GFP signal was not amplified using antibody staining. Scale bar: 5 µm. b Immunofluorescence images showing XPF–GFP LUD recruitment 1 h after 60 J/m² UVC through an 8-µm microporous filter in XPA-proficient and XPA KO U2OS cells treated with nontargeting siRNA (sictrl) or siRNA targeting FANCD2 (siFANCD2). Cells were immunostained against GFP and CPD, which was used as damage marker. Scale bar: 5 µm. c Quantification of XPF–GFP LUD recruitment in XPA-proficient and XPA KO cells, as determined by immunofluorescence experiments shown in b. The fold accumulation at sites of local damage was calculated over the nuclear background and plotted as average of at least 100 cells per condition from two independent experiments. Statistical significant difference (p < 0.05) is indicated by asterisk, n.s. non-significant. d Scatter plot of the log₂ SILAC ratio of the forward and reverse SILAC ERCC1–GFP immunoprecipitations experiments, comparing ERCC1–GFP interactors with and without UVC treatment (20 J/m², 1 h). UV-specific interactors are depicted in the upper right quadrant. In b error bars represent the SEM

Fig. 3

ICLR and NER core factors are recruited to psoralen-induced DNA damage. a Immunofluorescence images showing recruitment of endogenous FANCD2 and SLX4 to sites of local UVA laser microirradiation in untreated (laser) or psoralen-treated (8-MOP + laser, 50 µM 8-MOP, 2 h) U2OS cells. Cells were immunostained against FANCD2, SLX4 and γH2AX, which was used as damage marker. Scale bar: 5 µm. b Immunofluorescence images showing recruitment to sites of local UVA laser microirradiation in psoralen-treated U2OS cells (8-MOP + laser, 50 µM 8-MOP, 2 h) of endogenous XPA and XPF (left panels) and in psoralen-treated U2OS cells expressing ERCC–GFP or XPF–GFP (right panels). Cells were immunostained against XPA, XPF, GFP and γH2AX, which was used as damage marker. Scale bar: 5 µm

Fig. 4

ERCC1–XPF recruitment to psoralen-induced DNA damage is XPA and FANCD2 dependent. a Immunofluorescence images showing XPF–GFP recruitment to sites of local UVA laser microirradiation in psoralen-treated (50 µM 8-MOP, 2 h) XPA-proficient and XPA KO U2OS cells treated with nontargeting siRNA (sictrl) or siRNA targeting FANCD2 (siFANCD2). Cells were immunostained against GFP and γH2AX, which was used as damage marker. Scale bar: 5 µm. b Quantification of XPF–GFP recruitment to sites of local UVA laser microirradiation in psoralen-treated XPA-proficient and XPA KO U2OS cells, as determined by immunofluorescence experiments shown in a. The fold accumulation at sites of local damage was calculated over the nuclear background and plotted as average of at least 80 cells per condition from three independent experiments. Statistically significant difference (p < 0.05) is indicated by asterisk. c Immunofluorescence images showing ERCC1–GFP recruitment to sites of local UVA laser microirradiation in psoralen-treated (50 µM 8-MOP, 2 h) XPA-proficient and XPA KO U2OS cells treated with nontargeting siRNA (sictrl) or siRNA targeting FANCD2 (siFANCD2). Cells were stained against GFP and γH2AX, which was used as damage marker. Scale bar: 5 µm. d Quantification of ERCC1–GFP recruitment to sites of local UVA laser microirradiation in psoralen-treated XPA-proficient and XPA KO U2OS cells, as determined by immunofluorescence experiments shown in c. The fold accumulation at sites of local damage was calculated over the nuclear background and plotted as average of at least 100 cells per condition from two independent experiments. Statistically significant difference (p < 0.05) is indicated by asterisk. In b and d, error bars represent the SEM

Fig. 5

R689S and S786F mutant XPF is recruited to psoralen adducts in an XPA-dependent and FANCD2-independent manner. a Immunofluorescence images showing XPF recruitment to sites of local UVA laser microirradiation in psoralen-treated (50 µM 8-MOP, 2 h) U2OS XPF KO cells expressing GFP-tagged XPF carrying the amino acid mutations C236R, R689S or S786F, treated with nontargeting siRNA (sictrl) or siRNA targeting XPA (siXPA) or FANCD2 (siFANCD2). Cells were immunostained against GFP and γH2AX, which was used as damage marker. Scale bar: 5 µm. b Quantification of mutant XPF–GFP recruitment to sites of local UVA laser microirradiation in psoralen-treated U2OS XPF KO cells expressing GFP-tagged XPF carrying the amino acid mutations C236R, R689S or S786F, as determined by immunofluorescence experiments shown in a. The fold accumulation at sites of local damage was calculated over the nuclear background and plotted as average of at least 60 cells per condition from three independent experiments. Statistical significant difference (p < 0.05) compared to sicntrl of each cell line is indicated by asterisk. c Immunoblot analysis of cell lysate (input) and GFP immunoprecipitation samples (elute) from wild-type U2OS cells (U2OS) and XPF KO U2OS cells expressing GFP-tagged wild-type XPF (XPF–GFP) or XPF carrying the amino acid mutations R689S or S786F. Samples were analyzed with antibodies against GFP, ERCC1, SLX4, FANCD2, RPA70 and H2B (as loading control). Cells were mock treated (−) or incubated with 10 µg/ml MMC for 1 h before lysis. In the FANCD2 immunoblot, the asterisk indicates aspecific staining. In b error bars represent the SEM