Figure 4  TFIIH Functions and Mechanisms of XPD-Associated Disease Pleiotropy
(A) Cellular survival after UV irradiation. Rescue of hemizygous XpdTTD/KO survival by Xpd†XPCS and Xpd†XP alleles is illustrated by arrows marked A and B, respectively. UV survival of homozygous XpdXPCS/XPCS cells (asterisk) from the normally expressed viable allele (XpdXPCS) is depicted by a dotted line. Survival curves represent an average of four independent experiments; 1–2 cell lines per genotype were included in each experiment. Error bars indicate SEM between experiments.
(B) UV-UDS, a measure of global genome repair. Number of experiments: n = 15 (XpdTTD/TTD), n = 6 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS ), n = 2 (XpdTTD/†XP ); 1–2 cell lines per genotype were included in each experiment. The asterisk indicates significant difference with XpdTTD/TTD; crosses indicate significant differences with XpdTTD/KO.
(C) UV-RRS, a measure of transcription-coupled repair of UV-induced lesions. Number of experiments: n = 7 (XpdTTD/TTD), n = 2 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS ), n = 2 (XpdTTD/†XP ); 1–2 cell lines per genotype were included in each experiment.
(D) Incision/excision activity of combinations of altered TFIIH complexes in a reconstituted NER reaction. Equal amounts of single or mixed populations of recombinant TFIIHs (containing XPD, XPB, p62, p52, His-p44, Flag-p34, cdk7, cyclin H, Mat1, and p8) were mixed with recombinant XPG, XPF/ERCC1, XPC/hHR23B, RPA, and a radiolabelled synthetic NER substrate. The excision products (26–34 nucleotides in length) were visualised at nucleotide resolution on a denaturing polyacrylamide gel as indicated . Note the weak activity corresponding to each single and combined TFIIH complex (lanes 3–8) relative to the wt (lane 1) and negative controls (lane 2).
(E) Xpd dose-dependent reduction of TFIIH in homozygous XpdTTD/TTD, hemizygous XpdTTD/KO, and compound heterozygous XpdTTD/†XPCS and XpdTTD/†XP cells by comparative immunofluorescence of the p62 subunit of TFIIH. Roman numerals represent different microscopic slides and Arabic numerals different cell lines labelled as follows: (I) wt cells (1) labelled with 2-μm beads, XpdTTD/TTD cells (2) with 0.79-μm beads, and XpdTTD/KO cells (3) with no beads; (II) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XPCS cells (4) with no beads; and (III) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XP cells (5) with no beads.
(F) Quantification of immunofluorescent signal from at least 50 nuclei per cell line and 2–6 experiments per genotype. Bars representing cells analysed on the same microscopic slide are depicted side by side, with wt set at 100%. The p-value indicates minimum significant difference between wt and the indicated cell lines analysed on the same microscopic slide within one experiment.