The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently... more
The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with ther remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here we report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecula...
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Research Interests: Molecular Biology, Kinetics, Fluorescence Microscopy, DNA replication, Protein Dynamics, and 18 moreDNA repair, DNA, Cell line, Humans, Transcription Factor, Enzyme, Chromatin, Histones, Anthraquinones, Chromosome, PCNA, Physiological Stress Markers, Histone, Kinetic Parameter, DNA binding proteins, "Intercalating Agents", DNA marker, and Biochemistry and cell biology
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Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains... more
Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.
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In response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating... more
In response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating RNA metabolism. Here we discuss a replication-independent pathway activated by transcription-blocking DNA lesions, which utilizes the ATM signaling kinase to regulate spliceosome function in a reciprocal manner. We present a model according to which, displacement of co-transcriptional spliceosomes from lesion-arrested RNA polymerases, culminates in R-loop formation and non-canonical ATM activation. ATM signals in a feed-forward fashion to further impede spliceosome organization and regulates UV-induced gene expression and alternative splicing genome-wide. This reciprocal coupling between ATM and the spliceosome highlights the importance of ATM signaling in the cellular response to transcription-blocking lesions and supports a key role of the splicing...
Research Interests: Genetics and RNA biology
Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition,... more
Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing
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Research Interests: Materials Engineering, Condensed Matter Physics, Microscopy, Fluorescence Microscopy, Confocal Microscopy, and 10 moreDNA repair, Fluorescence Resonance Energy Transfer, Protein-protein interactions, Animals, FRET, Photobleaching, Nucleotide Excision Repair, DNA binding proteins, Spectral Imaging, and Biochemistry and cell biology
Research Interests: Radiation, Kinetics, Cancer, Fluorescence Microscopy, DNA replication, and 17 moreDNA repair, Cell Culture, DNA, In Vitro, Humans, Ultraviolet, Support, Enzyme, Cultured meat, Radiation Effect, Dose Response Relationship, UV irradiation, Monoclonal Antibody, Reference Values, Reference Value, Xeroderma Pigmentosum, and fibroblasts
Research Interests: Genetics, Adolescent, DNA damage, DNA repair, Transcription Factors, and 23 moreCell Culture, Transcription, Biological Sciences, DNA, Mental Retardation, Humans, Child, Skin Cancer, Mutation, Ultraviolet, Female, Cockayne syndrome, Male, American, Nucleotide Excision Repair, Gene, Transcription Factor, Gen, Sexual Development, Autosomal Recessive, DNA binding proteins, Xeroderma Pigmentosum, and Hair Diseases
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In this issue, Li et al. (2015) uncover roles for the XPB and XPD helicases and for XPA during damage verification in nucleotide excision repair, supporting a novel tripartite damage checking mechanism that combines extreme versatility... more
In this issue, Li et al. (2015) uncover roles for the XPB and XPD helicases and for XPA during damage verification in nucleotide excision repair, supporting a novel tripartite damage checking mechanism that combines extreme versatility with narrow specificity.
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The various symptoms associated with hereditary defects in the DNA damage response (DDR), which range from developmental and neurological abnormalities and immunodeficiency to tissue-specific cancers and accelerated aging, suggest that... more
The various symptoms associated with hereditary defects in the DNA damage response (DDR), which range from developmental and neurological abnormalities and immunodeficiency to tissue-specific cancers and accelerated aging, suggest that DNA damage affects tissues differently. Mechanistic DDR studies are, however, mostly performed in vitro, in unicellular model systems or cultured cells, precluding a clear and comprehensive view of the DNA damage response of multicellular organisms. Studies performed in intact, multicellular animals models suggest that DDR can vary according to the type, proliferation and differentiation status of a cell. The nematode Caenorhabditis elegans has become an important DDR model and appears to be especially well suited to understand in vivo tissue-specific responses to DNA damage as well as the impact of DNA damage on development, reproduction and health of an entire multicellular organism. C. elegans germ cells are highly sensitive to DNA damage induction and respond via classical, evolutionary conserved DDR pathways aimed at efficient and error-free maintenance of the entire genome. Somatic tissues, however, respond differently to DNA damage and prioritize DDR mechanisms that promote growth and function. In this mini-review, we describe tissue-specific differences in DDR mechanisms that have been uncovered utilizing C. elegans as role model.
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Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent... more
Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process.
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Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation... more
Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will...
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Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged... more
Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5 -end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into...
Research Interests: DNA damage, DNA repair, DNA, Nucleotide Excision Repair, DNA Structure, and 11 moreSecondary Structure, Protein Secondary Structure Prediction, Base Sequence, Recombinant Proteins, Xeroderma Pigmentosum, Nucleic Acid Conformation, Nucleotides, Biochemistry and cell biology, Molecular Structure, single stranded DNA, and Molecular Sequence Data
Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins... more
Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These fin...
Research Interests: Genetics, RNA, Fluorescence, DNA repair, Transcription Factors, and 30 moreGene expression, Sequence Analysis, In Situ Hybridization, Biological Sciences, DNA, Saccharomyces cerevisiae, Molecular, Phylogeny, Cell line, Environmental Sciences, Humans, Fluorescence in situ hybridization, Sequence alignment, Female, Animals, Male, Nucleotide Excision Repair, Nucleic Acids, Proteins, Cloning, Transcription Factor, Molecular cloning, HeLa cells, Amino Acid Profile, Amino Acid Sequence, Protein Binding, Chromosomes, DNA binding proteins, Gene Expression Regulation, and Molecular Sequence Data
TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine... more
TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.
Research Interests: Genetics, Fluorescence, DNA repair, Transcription Factors, Sequence Analysis, and 27 moreWestern blotting, In Situ Hybridization, Transcription, Biological Sciences, Saccharomyces cerevisiae, Molecular, Antibodies, Humans, Fluorescence in situ hybridization, Sequence alignment, Cockayne syndrome, Monoclonal Antibodies, Nucleotide Excision Repair, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Cloning, Transcription Factor, Gene Structure, Support, Molecular cloning, Electrophoresis, Monoclonal Antibody, Amino Acid Sequence, Polyacrylamide Gel, Chromosomes, Xeroderma Pigmentosum, Monoclonal, and Molecular Sequence Data
The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G)... more
The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. We report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associat...
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Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical... more
Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%-40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly-->arg change at amino acid 675 in the allele inherited from the patient's mother and a -1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in ...
Research Interests: Genetics, DNA damage, DNA repair, Transcription Factors, Cell Culture, and 18 moreBiological Sciences, Humans, Ultraviolet, Cockayne syndrome, Male, American, Nucleotide Excision Repair, Proteins, Radiation Effect, Genetic Disorder, UV light, Amino Acid Profile, Lymphocytes, Radiation Tolerance, DNA binding proteins, Xeroderma Pigmentosum, Child preschool, and fibroblasts
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The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that... more
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial ...
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The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair... more
The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda(-/-) ) mouse-model resembling TTD-A patients. Unexpectedly, Ttda(-/-) mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda(-/-) cells was not affected. Surprisingly, Ttda(-/-) cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. M...
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The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision... more
The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the ...
Research Interests: Cellular Biology, Kinetics, Fluorescent Dyes and Reagents, DNA damage, DNA repair, and 12 moreTranscription Factors, Biological Sciences, Business Management, Cell line, Molecular and cellular biology, Humans, Animals, Nucleotide Excision Repair, HeLa cells, fluorescence recovery after photobleaching (FRAP), Cell Survival, and DNA binding proteins
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Research Interests: Genetics, Fluorescence Microscopy, Confocal Microscopy, DNA repair, Single molecule, and 16 moreBiological Sciences, DNA, Environmental Sciences, Humans, Diffusion, DNA analysis, Nucleotide Excision Repair, Nucleic Acids, Protein Complex Detection, DNA binding, Reproducibility of Results, Protein Binding, Fusion Protein, DNA binding proteins, Nucleic Acid Conformation, and Nucleic Acid
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ABSTRACT
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Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living... more
Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response.