FIG. 2.

Inducible expression of wild-type p89 in XPB cells. (A) Expression of wild-type p89 can be induced by cadmium. HA-tagged wild-type p89 is under the control of a metal-inducible promoter. Wild-type p89 is detectable in XPB/wt-p89 cells even under steady-state conditions (lane 2) and can be induced by cadmium (lanes 3 to 8). No wild-type p89 is expressed even under high doses of cadmium in the XPB cell line (lane 1). (B) Quantification of p89 expression. A polyclonal antibody against the C terminus of p89 (SC-293) recognizes wild-type p89 but not truncated p89. Under steady-state conditions, XPB/wt-p89 cells express p89 to a level similar to that with the BJAB reference cell line. Probing for p62, another subunit of core TFIIH, shows similar levels of expression in all three cell lines, indicating that expressing wild-type p89 alters TFIIH quality but not protein turnover. (C) Quantification of p89 protein levels. XPB and XPB/wt-89 whole-cell lysates were subjected to Western blotting with α-p89 (Austral), which recognizes both truncated and wild-type p89. (D) Subcellular localization of wild-type p89 in XPB/wt-p89 cells. Wild-typep89 (detected by α-HA) is distributed in a finely dotted pattern throughout the nucleoplasm with relative sparing of nucleoli. Staining for cyclin H reveals colocalization with p89, indicating proper TFIIH assembly. Nuclei were counterstained with DAPI. (E) XPB/wt-p89 cells are DNA repair proficient. XPB cells, XPB/wt-p89 cells, XPB/wt-p89 cells under cadmium induction, and BJAB cells were UV irradiated with increasing doses, and survival rates (the ratio of treated cells/untreated cells) were assessed 4 days later. Low-dose UV irradiation dramatically reduced XPB cell survival, whereas XPB/wt-p89 cells were less sensitive. Comparison to a reference cell line (BJAB) indicates that the repair capacity is within normal range. Induction of wild-type p89 expression with 500 nM cadmium does not change survival compared to steady-state expression levels.