Targeting of the mouse gene Xpb. (A) Sequence of the last intron-exon border of the human XPB and XP11BE and the corresponding mouse Xpb alleles. C-to-A transversion (bold) creates a new splice acceptor site (CAG; underlined italics), resulting in a frame-shifted transcript and 40 novel amino acids at the C terminus derived from the frameshift (first five frameshifted amino acids are indicated). A mouse 4-bp insertion in mXpb creates a similar frameshift (Xpb^fs; bold). Due to differences in the nucleotide sequence between mice and humans, in mice the 4-bp insertion leads to substitution of the last 41 amino acids to 85 frame-shifted amino acids (G). (B) Schematic representation of the genomic structure and partial restriction map of the WT and targeted mouse Xpb loci. Black box, exon 15; p(A), polyadenylation signal; †, Xpb^fs 4-bp insertion. Probes A, B, and C are indicated with thick black lines. Restriction sites are abbreviated as follows: Nc, NcoI; Bg, BglII; B, BamHI; X, XbaI; C, ClaI. The diagnostic BglII site (Bg) introduced in the sequence is indicated in bold italics. (C) Southern blot analysis of NcoI- and BglII-digested genomic DNA from WT and Xpb^fs recombinant ES clones hybridized with probes A, C, and B as indicated. (D) Sequence of the last intron-exon border of mouse WT and Xpb^XPCS alleles. The WT splice acceptor site is underlined.Changed nucleotides in the mutated allele (bold) create an additional 5′ splice acceptor site (underlined) and stop codon (TAG; bold) in the altered reading frame only. Amino acids encoded by both WT and Xpb^XPCS reading frames are indicated. (E) Genomic structure of the Xpb WT and Xpb^XPCS alleles. The asterisk indicates the introduced 5′ splice acceptor site. (F) Southern blot analysis of NcoI-digested genomic DNA from WT ES cells (+/+), Xpb^XPCS recombinant ES clones (+/−), and homozygous Xpb^XPCS/XPCS mutant mice (−/−) hybridized with probes B and C. (G) Schematic representation of human (NP_000113.1) and mouse (NP_598419.1) XPB proteins derived from alleles depicted in panels A and D. NLS, nuclear localization signal; fs. aa, novel amino acids derived from the frameshift; the C-terminal recognition epitope of 2G12 MAb is indicated with a line. (H) Immunoblot analysis of whole-cell extracts from HeLa cells, two independent homozygous mutant Xpb^XPCS MEF lines (−/−), and WT MEFs (+/+). Note that the αXPB MAbs 1B3 and 2G12 recognize conserved epitopes within the N and C termini, respectively, and that the C-terminal epitope is absent in the truncated protein (G). The p62 subunit of TFIIH (stained with MAb 3C9; lower panel) served as qualitative control for loading. (I) Reduction of TFIIH protein levels in homozygous Xpb^XPCS primary MEFs visualized by comparative immunofluorescence assay. WT cells were labeled with 0.79-μm latex beads; Xpb^XPCS cells were unlabeled (asterisk). Left panel, phase contrast image; right panel, corresponding immunofluorescent image of the p62 subunit of TFIIH. Note the reduced signal in the Xpb^XPCS cells. (J) Quantification of the TFIIH level in Xpb^XPCS MEFs. The immunofluorescence signal from Xpb^XPCS cells was determined (at least 50 nuclei per genotype, two separate experiments with two independent Xpb^XPCS and WT cell lines) and expressed as the average percentage of the level in WT cells analyzed on the same microscopic slide. Error bars indicate the SEM between experiments. H.sp., Homo sapiens; M.m., Mus musculus.

Acute effects of UV-B on the skin of Xpb^XPCS mice. H&E staining of dorsal skin sections from shaven mice exposed to daily doses of 500 J/m² UVB for four consecutive days and sacrificed 1 week after the start of the treatment. Note the moderate epidermal hyperplasia, consisting of an increased number of cell layers (acanthosis, indicated with a bar) in the Xpb^XPCS skin and the much more severe effect in Xpa mice as evident from hyperemia (dilated capillaries filled with blood, indicated with a long arrow) and the absence of keratinized and epidermal layers (asterisk), reflecting severe “scaling of the skin.” Note the “swelling” of the nuclei after UV treatment in all genotypes compared to untreated controls (short arrows). Magnification, ×400.

Schematic representation of the relation between the extent of the DNA repair deficiency and the phenotypic variation. With a mild DNA repair defect, limited phenotypic variation is noted, whereas mice with a severe DNA repair defect die too fast to allow stochastic effects to manifest. The biggest phenotypic variation is expected in animals with an intermediate DNA repair defect. Lower panel: the existing mouse models are grouped from A to E based on phenotypic variation and presumed DNA repair (or other) defects. (A) Xpb^XPCS animals with a “mild” causative allele exhibit no detectable phenotypic deviation from the WT. (B) In mice mimicking the human “severe” CS, XPCS, or TTD causative mutations, phenotypes are relatively mild, suggesting that the DNA repair capacity is still proficient enough to battle most of the endogenous DNA damage load. (C) Xpa Xpb^XPCS mice display the widest phenotypic variation. On top of complete NER deficiency, these animals are also compromised in the removal of non-NER-type oxidative DNA lesions. Some symptoms in Xpa Xpb^XPCS mice appeared less penetrant (23% juvenile lethality; kyphosis and/or cachexia in 30% of animals) than others (tubular testicular atrophy was seen in all tested ageing males). Presumably, a random build-up of genome damage in the critical progenitor cells of individual mice may lead to diverse phenotypic consequences both during development and upon aging, depending on the number and tissue context of the affected cells. Xpa Xpd^TTD/XPCS mice were also placed within or near group C. Here the effect of the mutation is made “milder” by tissue-specific usage of each Xpd allele product and/or interallelic complementation. (D) Animals with NER progeria (Table 3) die nearly uniformly around weaning, leaving little time for stochastically accumulating lesions to manifest in phenotype. (E) Xpb^XPCS Xpd^TTD mice have defects in the repair of oxidative DNA damage (Fig. 2G and H) but may, in addition, have defects in activated transcription (18, 35), explaining their failure to survive beyond 1 to 2 days after birth (Table 3). Mouse models generated in this study are indicated (bold).

Cellular DNA repair characteristics. (A) UV-induced (15 J/m² UVC) unscheduled DNA repair synthesis capacity (UDS) of primary homozygous Xpb^XPCS MEFs. A representative experiment (three experiments total) is depicted. The P value indicates the significance of the difference between WT and Xpb^XPCS within the representative experiment. Error bars indicate the SEM. (B) Recovery of RNA synthesis after UV (10 J/m² UVC) irradiation (RRS). A representative experiment (three experiments total) is depicted. The P value indicates the significance of the difference between WT and Xpb^XPCS within the representative experiment. Error bars indicate the SEM. (C) UV survival curves averaged from four independent experiments. At least 2 cell lines per genotype were included. Error bars indicate SEM between experiments. (D) Gamma ray survival curves averaged from five independent experiments with two cell lines per genotype. Error bars indicate the SEM between experiments. (E) Reduction of TFIIH protein levels in Xpb^XPCS, Xpd^TTD single-mutant, and Xpb^XPCS Xpd^TTD double-mutant primary MEFs by comparative immunofluorescence analysis of p62 subunit of TFIIH. Quantification of the immunofluorescence signal is based on analysis of at least 50 nuclei per genotype in two separate experiments with two independent cell lines per genotype. WT cells are labeled with latex beads, mixed with the mutant cells, and cultured and immunostained on the same microscopic slide. Bars representing cell lines analyzed on the same microscopic slide are depicted side by side. The P value indicates the minimum significant difference between WT versus mutant cell lines analyzed on the same microscopic slide within one experiment. (F) UV survival curves averaged from four independent experiments. At least two cell lines per genotype were included. Error bars indicate the SEM between experiments. (G) Hypersensitivity of the indicated cells to acute oxidative damage. Gamma ray survival curves averaged from two independent experiments with two cell lines per genotype. Error bars indicate the SEM between experiments and lie within the symbol size. (H) Hypersensitivity of Xpb^XPCS Xpd^TTD and Xpa Xpb^XPCS cells to chronic oxidative injury. MEF cells of the indicated genotype were cultured in the continuous presence of the indicated concentration of paraquat for 3 days. Two cell lines per genotype were tested. For reasons of simplicity, on the depicted representative survival experiment, results from two independent cell lines for Xpb^XPCS and Xpa Xpb^XPCS cells were averaged and error bars depict the SEM between two independent cell lines within the given experiment. For the other genotypes in the given experiment, one cell line per genotype was used and error bars depict the SEM within the experiment.

Effects of additional NER mutations in Xpb^XPCS mice. (A) Kaplan-Meier survival curve of Xpa Xpb^XPCS mice. Shown are results for the WT (n = 36), Xpa (n = 34), Xpb^XPCS (n = 41), Xpa Xpb^XPCS (n = 30), and Xpb^XPCS Xpd^TTD (n = 5). Please note that for Xpb^XPCS Xpd^TTD mice, the exact time of death is unknown; it likely occurred within 1 to 2 days after birth (see Table 2 and above). (B) Body weight of Xpa Xpb^XPCS mice, plotted as a percentage of age and gender-matched littermate controls. Note the increase in body weight between 4.7 and 17 weeks. Error bars indicate the SEM. (C) Tail suspension test of 7-week-old male mice. The WT mouse depicted displays normal spreading of the hind limbs. Xpa Xpb^XPCS mice displayed heterogeneous behavior, ranging from normal to severe cramp-like seizures, spastic movements, and tremors of hind limbs as depicted here. Note also the smaller size of the Xpa Xpb^XPCS mouse. (D) Severe kyphosis in a 16-month-old male Xpa Xpb^XPCS mouse. (E) Premature testicular tubular atrophy of Xpa Xpb^XPCS mice. H&E-stained sections of the testis of 18-month and 7-week-old WT and Xpa Xpb^XPCS mice. Note the reduced thickness or absence of the germinal epithelium (bar), reduced occurrence of mature spermatids (arrow), and the increase in interstitial cells (asterisk) in Xpa Xpb^XPCS males at 18 months but not at 7 weeks. Magnification, ×100. (F) Photograph of a ∼24-h-old Xpb^XPCS Xpd^TTD double-mutant mouse and a double-heterozygote littermate. After apparent normal embryogenesis, double-mutant mice failed to grow and died within ∼36 h.