Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Transcription coupled DNA repair protein UVSSA binds to DNA and RNA: Mapping of nucleic acid interaction sites on human UVSSA.

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1-150) and a C-terminal (residues 495-605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606-662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

The molecular genetics of UV-Sensitive syndrome: A rare dermal anomaly.

Ultraviolet-sensitive syndrome is a rare skin disorder characterised by heterogeneous phenotypic spectrum of skin freckling, telangiectasia and acute sunburn. It usually has an autosomal recessive pattern. So far, only 18 patients from nine different families (Japanese, French, Israeli, Iranian and Pakistani) have been reported in scientific literature. Its precise prevalence is still unknown, but, according to an estimate, its prevalence ratio is 1:100,000 worldwide. Until now, only three genes have been reported to be involved in the syndrome; the Excision Repair Cross-Complementing, Group 6, the Excision Repair Cross-Complementing, Group 8 and the UV-Stimulated Scaffold Protein A (UVSSA). Among these genes, the last one is reported to be more prevalent among different ethnicities, including Pakistani. Physiologically, most of the syndrome genes are involved in the transcription-coupled nucleotide excision pathway. In order to reduce the disease severity, the patients are advised to use medicated skin moisturisers or sun-blocks, sunglasses and gloves, while going out in the sun to avoid sun exposure. The current narrative review was planned to discuss the molecular genetics and the mutational spectrum of the syndrome, and to describe the differential diagnosis of various related disorders in order to facilitate clinical researchers.

Generation of splice switching oligonucleotides targeting the Cockayne syndrome group B gene product in order to change the diseased cell state.

Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVSS), a disorder that causes mild symptoms. How the CSB gene determines a patient's fate is unknown, but one intriguing point is that in UVSS patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H2O2 treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells.

Understanding photodermatoses associated with defective DNA repair: Photosensitive syndromes without associated cancer predisposition.

Photodermatoses associated with defective DNA repair are a group of photosensitive hereditary skin disorders. In this review, we focus on diseases and syndromes with defective nucleotide excision repair that are not accompanied by an increased risk of cutaneous malignancies despite having photosensitivity. Specifically, the gene mutations and transcription defects, epidemiology, and clinical features of Cockayne syndrome, cerebro-oculo-facial-skeletal syndrome, ultraviolet-sensitive syndrome, and trichothiodystrophy will be discussed. These conditions may also have other extracutaneous involvement affecting the neurologic system and growth and development. Rigorous photoprotection remains an important component of the management of these inherited DNA repair-deficiency photodermatoses.

A Cockayne-Syndrome-Like Phenotype with a Homozygous Truncating UVSSA Variant: Might This Be a New Cause?

Introduction: UV-sensitive syndrome and Cockayne syndrome (CS) are rare autosomal recessive and transcription-coupled nucleotide excision repair disorders with different clinical manifestations, although some types are allelic. Case Presentation: We report on a patient who passed away at 15 years old with a progeroid-like appearance, cachexia, hearing loss, and dental anomalies, which led us to the diagnosis of Cockayne-like progeroid syndromes. Our clinical exome sequencing including all the known genes of progeroid syndromes revealed a homozygous stop-gain variant in the UVSSA gene. Conclusion: Although truncating variants in the UVSSA are known to cause UVsS3, their association with CS has not yet been defined. This case might be the first report of a CS-like phenotype caused by a defective UVSSA.

The awakening of DNA repair at Yale.

As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease.

Epigenomic signature of accelerated ageing in progeroid Cockayne syndrome.

Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.

Structural characterization of transcription-coupled repair protein UVSSA and its interaction with TFIIH protein.

UV-stimulated scaffold protein A (UVSSA) is a key protein in the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. UVSSA, an intrinsically disordered protein, interacts with multiple members of the pathway, tethering them into the complex. Several studies have reported that UVSSA recruits Transcription Factor IIH (TFIIH) via direct interaction, following which CSB is degraded and the lesion recognition TC-NER complex dissociates from the damage site to facilitate the DNA repair. Structural insights into these events remain largely unknown. Herein, we have investigated the interaction of human UVSSA with the Pleckstrin-Homology-domain of p62 subunit of TFIIH (p62-PHD) using biophysical techniques. We observed that UVSSA forms a stable complex with the p62-PHD in vitro. Small-angle scattering measurements using X-rays and neutrons revealed a significant change in pair-distance distribution function for UVSSA662/p62-PHD complex compared to UVSSA alone. Additionally, a significant decrease was observed in the radius of gyration of the complex. Our findings suggest that TFIIH binding to UVSSA causes significant conformational changes in UVSSA. We hypothesize that these conformational changes play an important role in the dissociation of the lesion recognition TC-NER complex.

A Homozygous Nonsense Variant in UVSSA Causes UV-sensitive Syndrome from Very Large Kindred: The First Report from Iran.

Background: Recessive disruptive mutations in nucleotide excision repair genes are responsible for a wide range of cutaneous photosensitivity and, in some cases, are associated with multi-system involvement. The heterogeneous nature of these conditions makes next-generation sequencing the method of choice to detect disease-causing variants. Materials and Methods: A patient from a large multiplex inbred Iranian kindred with several individuals suffering from skin sun-sensitive manifestations underwent complete clinical and molecular evaluations. Whole exome sequencing (WES) was performed on the genomic sample of the proband, followed by bioinformatics analysis. Subsequently, co-segregation of the candidate variant with the condition was performed by Sanger sequencing. Results: A rare homozygous nonsense variant, c.1040G>A (p. Trp347*), was identified in the UVSSA gene, resulting in UV-sensitive syndrome (UVSS) complementation group A. The global minor allele frequency of the variant is < 0.001 in population databases. Tryptophan 347 residue is conserved among mammalians and vertebrates, and the null variant is believed to lead to a truncated protein with cellular mislocalization. Conclusions: Here, we report the first genetic diagnosis of UVSS-A in Iran via the successful application of Next-generation sequencing, which expands our understanding of the molecular pathogenesis of this condition.

UV-sensitive syndrome: Whole exome sequencing identified a nonsense mutation in the gene UVSSA in two consanguineous pedigrees from Pakistan.

BACKGROUND: UV-sensitive syndrome (UVSS) is a rare autosomal recessive genodermatosis characterised by photosensitivity, and hyperpigmentation, freckling, and dryness of sun exposed areas. In contrast to other photosensitivity disorders, affected patients show no predisposition to cutaneous melanoma or neurological dysfunction. UVSS results from a defect in the transcription-coupled nucleotide excision repair (TC-NER) mechanism. UVSS can be caused by mutations in the genes ERCC8, ERCC6, and UVSSA. OBJECTIVE: To determine the underlying genetic cause of UVSS and its functional consequences in nine members of two large, unrelated consanguineous pedigrees from Pakistan. METHODS: Genomic DNA from one affected member of each family was subjected to whole exome sequencing. The identified mutation was then validated via Sanger sequencing using samples from all available family members. Molecular cloning and mammalian cell cultures were used for the translation and localisation of wild type (WT) and mutant constructs. RESULTS: A novel homozygous nonsense mutation, (c.1040G>A [p.(Trp347*)]), was detected in exon 6 of the UVSSA gene in both families. Sanger sequencing revealed co-segregation of the nonsense mutation with the UVSS phenotype. Immunoblotting revealed the anticipated 81kDa band for the WT construct, and a truncated protein of around 39kDa for the mutant. In mutant samples, immunofluorescence revealed mislocalisation of UVSSA from the nucleus to the cytoplasm. CONCLUSIONS: This is the first report of UVSS in the Pakistani population and the fourth report of a disease-causing mutation in UVSSA. The study broadens the UVSSA mutational spectrum, and contributes to functional understanding of truncated UVSSA proteins.

Inhibition of UVSSA ubiquitination suppresses transcription-coupled nucleotide excision repair deficiency caused by dissociation from USP7.

Transcription-coupled nucleotide excision repair (TC-NER) is a subpathway of nucleotide excision repair that efficiently removes transcription-blocking DNA damage from the transcribed strands of active genes. UVSSA is a causative gene for UV-sensitive syndrome (UVS S), which is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in TC-NER. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, forms a complex with ubiquitin-specific peptidase 7 (USP7) and is stabilized by interaction with USP7. The central region of UVSSA, which contains the tumor necrosis factor receptor-associated factor (TRAF)-binding motif, is required for the interaction with the N-terminal TRAF domain of USP7. Here, we showed that UVSSA is mono-ubiquitinated in vitro and identified a lysine residue (Lys414 ) in UVSSA as the target of ubiquitination. The deubiquitination activity of USP7 was inhibited by the ubiquitin-conjugating enzyme UbcH6. Lys414 was also modified by poly-ubiquitin chains in vivo. UVSSA deficient in the interaction with USP7 is ubiquitinated and degraded by the proteasome, and the degradation leads to deficiency in TC-NER. The substitution of Lys414 by Arg of UVSSA inhibited its degradation and thereby suppressed the deficiency in TC-NER.