Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

Abstract

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.

Figure 1

Reduced repair of photoproducts in XP-E cells. XP-E cells were treated with UVC (10 J/m²) and incubated for up to 24 h. The removal of (A) 6-4PP and (B) CPD was measured with an ELISA assay. Results represent mean values ± sem of at least n=3 experiments. Normal cells (AG13145) - closed diamonds ◆; XPD cells: XP408BE –open squares □, XP37BE – open vertical triangles ▵, XP66BE – open inverted triangles ▽, XP1GO – open circles ○.

Figure 2

Recruitment and redistribution of NER proteins after focal UV irradiation in normal and XP-E (XP37BE) cells. Normal cells (0.8 μm beads – red arrows) and XP-E cells (2 μm beads – yellow arrows) were co-cultured and irradiated with 100 J/m² UVC, fixed, permeabilized, and double stained with antibodies specific for XP proteins along with antibodies for CPD or for 6-4PP at different time points after irradiation. Cells were visualized with Alexa Fluor R 568 goat anti-mouse IgG conjugate. (A) Time course of XPC, CPD and 6-4PP recruitment and redistribution. (B) Localization of XPB, XPG, XPA and XPF proteins at 0.1 h and 0.5 h after focal UV.

Figure 3

Schematic diagram of early and late global genome nucleotide excision repair in cells from normal and XP-E patients (see text for details).