An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A.

DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.

The ubiquitin receptor Rad23: at the crossroads of nucleotide excision repair and proteasomal degradation.

A protein that exemplifies the intimate link between the ubiquitin/proteasome system (UPS) and DNA repair is the yeast nucleotide excision repair (NER) protein Rad23 and its human orthologs hHR23A and hHR23B. Rad23, which was originally identified as an important factor involved in the recognition of DNA lesions, also plays a central role in targeting ubiquitylated proteins for proteasomal degradation, an activity that it shares with other ubiquitin receptors like Dsk2 and Ddi1. Although the finding that Rad23 serves as a ubiquitin receptor explains to a large extent its importance in proteasomal degradation, the precise mode of action of Rad23 in NER and the possible link with the UPS is less clear. In this review, we discuss our present knowledge on the functions of Rad23 in protein degradation and DNA repair and speculate on the importance of the dual roles of Rad23 for the cell's ability to cope with stress conditions.

UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells.

Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery.

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells.

Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER.

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.

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions.

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.

C-terminal UBA domains protect ubiquitin receptors by preventing initiation of protein degradation.

The ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC.

To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in the nucleus under genotoxic stress conditions.

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure.

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.

DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A.

Chromatin changes within the context of DNA repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional nucleotide excision repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM- and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA repair-induced chromatin remodeling.

Mathematical modeling of nucleotide excision repair reveals efficiency of sequential assembly strategies.

Nucleotide excision repair (NER) requires the concerted action of many different proteins that assemble at sites of damaged DNA in a sequential fashion. We have constructed a mathematical model delineating hallmarks and general characteristics for NER. We measured the assembly kinetics of the putative damage-recognition factor XPC-HR23B at sites of DNA damage in the nuclei of living cells. These and other in vivo kinetic data allowed us to scrutinize the dynamic behavior of the nucleotide excision repair process in detail. A sequential assembly mechanism appears remarkably advantageous in terms of repair efficiency. Alternative mechanisms for repairosome formation, including random assembly and preassembly, can readily become kinetically unfavorable. Based on the model, new experiments can be defined to gain further insight into this complex process and to critically test model predictions. Our work provides a kinetic framework for NER and rationalizes why many multiprotein processes within the cell nucleus show sequential assembly strategy.

TFIIH plays an essential role in RNA polymerase I transcription.

TFIIH is a multisubunit protein complex that plays an essential role in nucleotide excision repair and transcription of protein-coding genes. Here, we report that TFIIH is also required for ribosomal RNA synthesis in vivo and in vitro. In yeast, pre-rRNA synthesis is impaired in TFIIH ts strains. In a mouse, part of cellular TFIIH is localized within the nucleolus and is associated with subpopulations of both RNA polymerase I and the basal factor TIF-IB. Transcription systems lacking TFIIH are inactive and exogenous TFIIH restores transcriptional activity. TFIIH is required for productive but not abortive rDNA transcription, implying a postinitiation role in transcription. The results provide a molecular link between RNA polymerase I transcription and transcription-coupled repair of active ribosomal RNA genes.

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place primarily at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.

Rapid switching of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo.

The transcription/repair factor TFIIH operates as a DNA helix opener in RNA polymerase II (RNAP2) transcription and nucleotide excision repair. To study TFIIH in vivo, we generated cell lines expressing functional GFP-tagged TFIIH. TFIIH was homogeneously distributed throughout the nucleus with nucleolar accumulations. We provide in vivo evidence for involvement of TFIIH in RNA polymerase I (RNAP1) transcription. Photobleaching revealed that TFIIH moves freely and gets engaged in RNAP1 and RNAP2 transcription for approximately 25 and approximately 6 s, respectively. TFIIH readily switches between transcription and repair sites (where it is immobilized for approximately 4 min) without large-scale alterations in composition. Our findings support a model of diffusion and random collision of individual components that permits a quick and versatile response to changing conditions.