The XPA-binding domain of ERCC1 is required for nucleotide excision repair but not other DNA repair pathways

Abstract

The endonuclease ERCC1-XPF incises the damaged strand of DNA 5' to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1(N110A/Y145A), the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1(N110A/Y145A) protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin.

FIGURE 1.

Structure of an XPA peptide bound to the central domain of ERCC1. ERCC1 is shown in gray, and the XPA peptide with the highly conserved residues 72–75 (GGGF) is shown in purple. Residues selected for site-directed mutagenesis in ERCC1 (Asn-110, Tyr-145, and Tyr-152) are highlighted in atom color. The picture is adapted from Ref. .

FIGURE 2.

Mutations in the XPA-binding domain of ERCC1 affect NER activity but not nuclease activity in vitro. A, incision of a stem loop substrate by wild-type and mutant ERCC1-XPF. A 5′-³²P-labeled stem-loop DNA substrate (6.7 nm) was incubated with 6.7 nm (lanes 2, 4, 6, 8, 10, and 12) or 26.8 nm (lanes 3, 5, 7, 9, 11, and 13) ERCC1-XPF in the presence of 0.4 mm MnCl₂. B, NER activity of wild-type and mutant ERCC1-XPF. A plasmid containing a site-specific 1,3-intrastrand cisplatin DNA cross-link (50 ng) was incubated with a whole cell extract from ERCC1-XPF-deficient cells (XP2YO) complemented with recombinant ERCC1-XPF containing the indicated mutations in ERCC1 (N110A, Y145A, Y152A) or XPF (D720A). The excised DNA fragments of 24–32 nucleotides were detected by annealing a complementary oligonucleotide containing a non-complementary 4G overhang and filling in with [α-³²P]dCTP. Protein concentrations of ERCC1-XPF were 13.4 nm (lanes 2, 4, 6, 8, 10, 12, and 14) and 53.6 nm (lanes 3, 5, 7, 9, 11, 13, and 15). A labeled low molecular weight DNA ladder (New England Biolabs) was used as a marker. The position of a 25-mer is indicated.

FIGURE 3.

Mutations in the XPA-binding domain of ERCC1 affect its recruitment to sites of UV damage. A, expression levels of ERCC1 in transduced UV20 cells. Transduced cells express human ERCC1 tagged with hemagglutinin. Note that human ERCC1 has a slower mobility than the CHO protein due to larger size (297 amino acids versus 293 amino acids) and the presence of the hemagglutinin tag. Tubulin was used as a loading control. B, ERCC1-deficient CHO cells were transduced with wild-type or mutant ERCC1 and irradiated with UV light (120 J/m²) through a polycarbonate filter with 5-μm pores and then fixed and stained for ERCC1 (green) and (6-4)PP (red). DAPI, 4′-6′-diamino-2-phenylindole. C, graphical representation of the percentage of co-localization of ERCC1 with (6-4)PP in UV20 cells expressing various mutants of ERCC1. Data represent the average of at least three independent experiments ± S.D. (error bars). 100 cells were counted for each experiment.

FIGURE 4.

UV damage persists in UV20 cells expressing ERCC1^N110A/Y145A but not wild-type ERCC1. A, untransduced UV20 cells or cells expressing wild-type ERCC1 or ERCC1^N110A/Y145A were UV-irradiated as described in the legend for Fig. 3, cultured for 0, 1, 2, 4, 8, or 24 h following UV irradiation, and then fixed and stained for (6-4)PP. B, graphic representation of the percentage of cells with persistent (6-4)PP at various time points. Data represent the average of at least three independent experiments ± S.D. (error bars). 100 cells were counted for each experiment.

FIGURE 5.

Mutations in the XPA-binding domain of ERCC1 inhibit NER but not ICL or DSB repair. A–D, clonogenic survival assays to measure the sensitivity of CHO cell lines AA8 (WT) (red diamonds), Ercc1^−/− UV20 (blue diamonds), and UV20 expressing ERCC1^WT (green diamonds), ERCC1^N110A (black diamonds), ERCC1^Y145A (purple diamonds), or ERCC1^N110A/Y145A (yellow diamonds) to UV-C (A), mytomycin C (MMC) (B), cisplatin (C), or ionizing radiation (D). The data are plotted as the percentage of colonies that grew on the treated plates relative to untreated plates ± S.E. (error bars). cDDP, cis-diamminedichloroplatinum(II).