Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles

Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of “null” alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals

TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription

The general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. Here the authors provide evidence that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module within the preinitiation complex before their release during transcription

Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC–hHR23B, XPG, and ERCC1–XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC–hHR23B, XPG, and ERCC1–XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase δ or ε, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins

SerpinB2 is involved in cellular response upon UV irradiation

Abstract Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.clos

Rescue of TTD-Associated Segmental Progeroid Features in Compound Heterozygous <i>Xpd^TTD/†XPCS</i> Mice

<div><p>(A) Photographs of 20-mo-old wt, compound heterozygous <i>Xpd^TTD/†XPCS,</i> and homozygous <i>Xpd^TTD/TTD</i> mice. Note the extreme cachexia (lack of subcutaneous fat) in the <i>Xpd^TTD/TTD</i> mouse and the absence of this phenotype in wt and <i>Xpd^TTD/†XPCS</i> mice.</p> <p>(B) Radiographs of 20-mo-old male wt, <i>Xpd^TTD/†XPCS,</i> and <i>Xpd^TTD/TTD</i> mice. Ageing <i>Xpd^TTD/TTD</i> mice develop kyphosis (curvature of the spinal column) and reduction of bone mineral density as shown in the 6–8 segment of the tail vertebrae counted from the pelvis (see close-up at right). Note the absence of these features in the <i>Xpd^{TTD / † XPCS}</i> mouse.</p> <p>(C) Quantification of relative bone mineral density of tail vertebrae from 20-mo-old male wt (<i>n</i> = 3), <i>Xpd^TTD/†XPCS</i> (<i>n</i> = 4), and <i>Xpd^TTD/TTD</i> (<i>n</i> = 3) mice. The <i>p</i>-values indicate the significance of the difference relative to <i>Xpd^TTD/TTD</i>. Error bars indicate SEM.</p> <p>(D) Body weight curves as a function of time. Note that the age-dependent cachexia observed in <i>Xpd^TTD/TTD</i> mice was rescued in both male and female <i>Xpd^{TTD / †XPCS}</i> mice. Significant differences between wt and <i>Xpd^TTD/TTD</i> but not between wt and <i>Xpd^TTD/†XPCS</i> mice were observed at 9 and 18 mo of age as indicated by asterisks. Error bars indicate SEM.</p></div

TFIIH Functions and Mechanisms of XPD-Associated Disease Pleiotropy

<div><p>(A) Cellular survival after UV irradiation. Rescue of hemizygous <i>Xpd^TTD/KO</i> survival by <i>Xpd^†XPCS</i> and <i>Xpd^†XP</i> alleles is illustrated by arrows marked A and B, respectively. UV survival of homozygous <i>Xpd^XPCS/XPCS</i> cells (asterisk) from the normally expressed viable allele <i>(Xpd^XPCS)</i> 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.</p> <p>(B) UV-UDS, a measure of global genome repair. Number of experiments: <i>n</i> = 15 <i>(Xpd^TTD/TTD), n</i> = 6 <i>(Xpd^TTD/KO), n</i> = 4 <i>(Xpd^TTD/†XPCS</i><i>)</i>, <i>n</i> = 2 <i>(Xpd^TTD/†XP</i><i>);</i> 1–2 cell lines per genotype were included in each experiment. The asterisk indicates significant difference with <i>Xpd^TTD/TTD;</i> crosses indicate significant differences with <i>Xpd^TTD/KO</i>.</p> <p>(C) UV-RRS, a measure of transcription-coupled repair of UV-induced lesions. Number of experiments: <i>n</i> = 7 <i>(Xpd^TTD/TTD), n</i> = 2 <i>(Xpd^TTD/KO), n</i> = 4 <i>(Xpd^TTD/†XPCS</i><i>)</i>, n = 2 <i>(Xpd^TTD/†XP</i><i>)</i>; 1–2 cell lines per genotype were included in each experiment.</p> <p>(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).</p> <p>(E) <i>Xpd</i> dose-dependent reduction of TFIIH in homozygous <i>Xpd^TTD/TTD,</i> hemizygous <i>Xpd^TTD/KO,</i> and compound heterozygous <i>Xpd^TTD/†XPCS</i> and <i>Xpd^TTD/†XP</i> 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, <i>Xpd^TTD/TTD</i> cells (2) with 0.79-μm beads, and <i>Xpd^TTD/KO</i> cells (3) with no beads; (II) wt cells (1) labelled with 0.79-μm beads and <i>Xpd^TTD/†XPCS</i> cells (4) with no beads; and (III) wt cells (1) labelled with 0.79-μm beads and <i>Xpd^TTD/†XP</i> cells (5) with no beads.</p> <p>(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 <i>p</i>-value indicates minimum significant difference between wt and the indicated cell lines analysed on the same microscopic slide within one experiment.</p></div