http://repub.eur.nl/res/aut/4482/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33749/ 2011-09-01T00:00:00Z Trichothiodystrophy (TTD) is a rare autosomal premature-ageing and neuroectodermal disease. The photohypersensitive form of TTD is caused by inherited mutations in three of the 10 subunits of the basal transcription factor TFIIH. TFIIH is an essential transcription initiation factor that is also pivotal for nucleotide excision repair (NER). Photosensitive TTD is explained by deficient NER, dedicated to removing UV-induced DNA lesions. TTD group A (TTD-A) patients carry mutations in the smallest TFIIH subunit, TTDA, which is an 8-kDa protein that dynamically interacts with TFIIH. TTD-A patients display a relatively mild TTD phenotype, and TTD-A primary fibroblasts exhibit moderate UV sensitivity despite a rather low level of UV-induced unscheduled DNA synthesis (UDS). To investigate the rationale of this seeming discrepancy, we studied the repair kinetics and the binding kinetics of TFIIH downstream NER factors to damaged sites in TTD-A cells. Our results show that TTD-A cells do repair UV lesions, although with reduced efficiency, and that the binding of downstream NER factors on damaged DNA is not completely abolished but only retarded. We conclude that in TTD-A cells repair is not fully compromised but only delayed, and we present a model that explains the relatively mild photosensitive phenotype observed in TTD-A patients. http://repub.eur.nl/res/pub/28519/ 2010-07-01T00:00:00Z In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular and molecular biology, enabling the localization of proteins within cellular compartments to be analysed and to determine kinetic parameters of enzymatic reactions in living nuclei to be measured. Recently, in vivo DNA labelling by DNA-stains such as DRAQ5, has provided the opportunity to measure kinetic reactions of GFP-fused proteins in targeted areas of the nucleus with different chromatin compaction levels. To verify the suitability of combining DRAQ5-staining with protein dynamic measurements, we have tested the cellular consequences of DRAQ5 DNA intercalation. We show that DRAQ5 intercalation rapidly modifies both the localization and the mobility properties of several DNA-binding proteins such as histones, DNA repair, replication and transcription factors, by stimulating a release of these proteins from their substrate. Most importantly, the effect of DRAQ5 on the mobility of essential cellular enzymes results in a potent inhibition of the corresponding cellular functions. From these observations, we suggest that great caution must be used when interpreting live cell data obtained using DRAQ5. http://repub.eur.nl/res/pub/28259/ 2010-06-11T00:00:00Z Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSBdel) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSBdelremains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process. http://repub.eur.nl/res/pub/24972/ 2009-10-01T00:00:00Z Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies. http://repub.eur.nl/res/pub/24288/ 2009-09-25T00:00:00Z Recent studies have reported that elevated levels of galectin-7 in different types of cancer. The mechanisms underlying its abnormal regulation in cancer cells remain, however, unknown. Here, we have examined the relationship between galectin-7 and p53, a gene previously associated with upregulation of galectin-7. While RNA and protein analyses revealed a consistent and irreversible upregulation of galectin-7 throughout progression of lymphoma, no correlation with p53 was found. In fact, most of the lymphoma cell lines expressing high levels of galectin-7 did not express any detectable level of p53, although expressed p21Waf1/Cip1gene following doxorubicin treatment, indicating that p53 was functional in these cells. Methylation-specific polymerase chain reaction (MS-PCR) analyses rather suggested that galectin-7 expression was associated with changes in DNA methylation. This conclusion was supported by data using demethylating agent 5-aza-dC. Furthermore, disruption of the DNA methylases dnmt1 and dnmt3a induced galectin-7. Collectively, our data suggest that abnormal expression of galectin-7 in lymphoma cells is not dependent on p53, but is rather associated with DNA hypomethylation. http://repub.eur.nl/res/pub/24555/ 2009-04-22T00:00:00Z Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1-XPF and 3′ to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5′ incision by ERCC1-XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a cut-patch-cut-patch mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity. http://repub.eur.nl/res/pub/29534/ 2008-12-01T00:00:00Z Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UVactivated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions. Copyright http://repub.eur.nl/res/pub/30196/ 2008-12-01T00:00:00Z Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA. http://repub.eur.nl/res/pub/35561/ 2007-03-01T00:00:00Z 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. http://repub.eur.nl/res/pub/14006/ 2006-06-01T00:00:00Z 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. http://repub.eur.nl/res/pub/3220/ 2004-07-01T00:00:00Z 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.