Cockayne Syndrome Proteins Research Articles

The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage.

Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates.

Loss of Proteostasis Is a Pathomechanism in Cockayne Syndrome

Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS.

TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA–DDB1–CUL4A–RBX1 cullin–RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC’s binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.

Initial brain aging: heterogeneity of mitochondrial size is associated with decline in complex I-linked respiration in cortex and hippocampus

Brain aging is accompanied by declining mitochondrial respiration. We hypothesized that mitochondrial morphology and dynamics would reflect this decline. Using hippocampus and frontal cortex of a segmental progeroid mouse model lacking Cockayne syndrome protein B (CSBm/m) and C57Bl/6 (WT) controls and comparing young (2–5 months) to middle-aged mice (13–14 months), we found that complex I-linked state 3 respiration (CI) was reduced at middle age in CSBm/m hippocampus, but not in CSBm/m cortex or WT brain. In hippocampus of both genotypes, mitochondrial size heterogeneity increased with age. Notably, an inverse correlation between heterogeneity and CI was found in both genotypes, indicating that heterogeneity reflects mitochondrial dysfunction. The ratio between fission and fusion gene expression reflected age-related alterations in mitochondrial morphology but not heterogeneity. Mitochondrial DNA content was lower, and hypoxia-induced factor 1α mRNA was greater at both ages in CSBm/m compared to WT brain. Our findings show that decreased CI and increased mitochondrial size heterogeneity are highly associated and point to declining mitochondrial quality control as an initial event in brain aging.

NAP1L1 accelerates activation and decreases pausing to enhance nucleosome remodeling by CSB.

Cockayne syndrome protein B (CSB) belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, and CSB is the only ATP-dependent chromatin remodeler essential for transcription-coupled nucleotide excision DNA repair. CSB alone remodels nucleosomes ∼10-fold slower than the ACF remodeling complex. Strikingly, NAP1-like histone chaperones interact with CSB and greatly enhance CSB-mediated chromatin remodeling. While chromatin remodeling by CSB and NAP1-like proteins is crucial for efficient transcription-coupled DNA repair, the mechanism by which NAP1-like proteins enhance chromatin remodeling by CSB remains unknown. Here we studied CSB's DNA-binding and nucleosome-remodeling activities at the single molecule level in real time. We also determined how the NAP1L1 chaperone modulates these activities. We found that CSB interacts with DNA in two principle ways: by simple binding and a more complex association that involves gross DNA distortion. Remarkably, NAP1L1 suppresses both these interactions. Additionally, we demonstrate that nucleosome remodeling by CSB consists of three distinct phases: activation, translocation and pausing, similar to ACF. Importantly, we found that NAP1L1 promotes CSB-mediated remodeling by accelerating both activation and translocation. Additionally, NAP1L1 increases CSB processivity by decreasing the pausing probability during translocation. Our study, therefore, uncovers the different steps of CSB-mediated chromatin remodeling that can be regulated by NAP1L1.

061 Premature aging in Cockayne syndrome due to a failure in ribosomal biogenesis?

061 Premature aging in Cockayne syndrome due to a failure in ribosomal biogenesis? M Alupei, P esser, K Scharffetter-Kochanek and S Iben 1 University of Ulm, Ulm, Germany and 2 University of freiburg, Freiburg, Germany Cockayne syndrome (CS) is a progeroid disease characterized by growth failure, neurological degeneration, loss of subcutaneous fat and cachexia. The mutations of this polygenic disease inactivate a subbranch of the DNA-repair pathway nucleotide excision repair (NER), thus Cockayne syndrome is regarded as a DNA-repair disease. As the total loss of DNA repair by the NER in xeroderma pigmentosum does not lead to childhood degeneration, alternative functions of the CS proteins might account for the disease. One common alternative function of the Cockayne syndrome proteins is the key step in ribosomal biogenesis, transcription by RNA polymerase I in the nucleolus. We investigated the cellular consequences of a failure of RNA polymerase I transcription in CS-cells and discovered a circulus vitiosus repressing ribosomal biogenesis. A disturbed RNA polymerase I transcription gives rise to ribosomes with a low translational fidelity that produce misfolded proteins. Misfolded proteins provoke endoplasmic reticulum stress and an unfolded protein response that represses RNA polymerase I transcription. The oxidative hypersensitivity of Cockayne syndrome cells might be due to oxidation of misfolded proteins. The elevated apoptosis rate of CS cells after oxidative challenge can be overcome by pharmacological chaperones that dampen endoplasmic reticulum stress and the unfolded protein response. Pharmacological chaperones are restoring proper RNA polymerase I transcription by de-repression thus breaking the circulus vitiosus. This might be a treatment opportunity for children suffering from premature aging. Moreover, loss of proteostasis due to a reduced translational fidelity of ribosomes might be a mechanism contributing to aging and aging associated diseases in humans. 062 Stress perception impacts on clinical signs of skin ageing and modifies the epigenome R Pattinson, C Bundy, L Cordingley, S Kyle, W Yeung, G Beauchef, A Nkengne, A O’Callaghan, CE Griffiths and RE Watson 1 University of Manchester, Manchester, United Kingdom, 2 Laboratoire CLARINS, Pontoise, France and 3 Fios Genomics Ltd., Edinburgh, United Kingdom In this study we tested the commonly held assumption that stress affects the appearance of skin through the premature occurrence of clinical features of skin ageing (lines, wrinkles and dark under-eye circles). We recruited 84 healthy adult female volunteers (age range, 24 e 40 years; mean 30.7 4.7 years); each volunteer completed a comprehensive battery of psychological and sleep questionnaires (State-Trait Anxiety Inventory, Perceived Stress Score, Hospital Anxiety and Depression Scale, Pittsburgh sleep quality index [PSQI], Ford insomnia response to stress test [FIRST] and consensus sleep diary), clinical photography (VISIA-CR ) and provided a sample of whole blood for epigenetic analysis. Image analysis of clinical photographs allowed the objective quantification of wrinkles around the eye (eye bag and crow’s feet), the nasolabial fold and deep mid-forehead frown lines. Initial analysis correlated perceived stress with worse sleep patterns; those volunteers who perceived stress more keenly had significantly disturbed sleep as measured by PSQI and FIRST with reduced sleep efficiency (r1⁄4-0.22; P<0.05) and increased wakefulness after sleep onset (r1⁄40.32; P<0.01). In addition, sleep onset latency positively correlated with increased measures for nasolabial folds, frown lines and crow’s feet wrinkles (r1⁄4-0.47; p<0.01). We next stratified the volunteers on the basis of their stress perception response to identify extreme groups (high stress response, n1⁄412; low stress response, n1⁄412) for epigenetic analysis of whole blood. Using Infinium HumanMethylation450BeadChip, principal component analysis identified significant clustering of methylation signatures between the two groups, with 484 differentially methylated regions observed (P<0.001). Hence, how an individual perceives psychological stress appears to impact on physical signs of skin ageing although the precise mechanism remains unclear.

062 Stress perception impacts on clinical signs of skin ageing and modifies the epigenome

061 Premature aging in Cockayne syndrome due to a failure in ribosomal biogenesis? M Alupei, P esser, K Scharffetter-Kochanek and S Iben 1 University of Ulm, Ulm, Germany and 2 University of freiburg, Freiburg, Germany Cockayne syndrome (CS) is a progeroid disease characterized by growth failure, neurological degeneration, loss of subcutaneous fat and cachexia. The mutations of this polygenic disease inactivate a subbranch of the DNA-repair pathway nucleotide excision repair (NER), thus Cockayne syndrome is regarded as a DNA-repair disease. As the total loss of DNA repair by the NER in xeroderma pigmentosum does not lead to childhood degeneration, alternative functions of the CS proteins might account for the disease. One common alternative function of the Cockayne syndrome proteins is the key step in ribosomal biogenesis, transcription by RNA polymerase I in the nucleolus. We investigated the cellular consequences of a failure of RNA polymerase I transcription in CS-cells and discovered a circulus vitiosus repressing ribosomal biogenesis. A disturbed RNA polymerase I transcription gives rise to ribosomes with a low translational fidelity that produce misfolded proteins. Misfolded proteins provoke endoplasmic reticulum stress and an unfolded protein response that represses RNA polymerase I transcription. The oxidative hypersensitivity of Cockayne syndrome cells might be due to oxidation of misfolded proteins. The elevated apoptosis rate of CS cells after oxidative challenge can be overcome by pharmacological chaperones that dampen endoplasmic reticulum stress and the unfolded protein response. Pharmacological chaperones are restoring proper RNA polymerase I transcription by de-repression thus breaking the circulus vitiosus. This might be a treatment opportunity for children suffering from premature aging. Moreover, loss of proteostasis due to a reduced translational fidelity of ribosomes might be a mechanism contributing to aging and aging associated diseases in humans. 062 Stress perception impacts on clinical signs of skin ageing and modifies the epigenome R Pattinson, C Bundy, L Cordingley, S Kyle, W Yeung, G Beauchef, A Nkengne, A O’Callaghan, CE Griffiths and RE Watson 1 University of Manchester, Manchester, United Kingdom, 2 Laboratoire CLARINS, Pontoise, France and 3 Fios Genomics Ltd., Edinburgh, United Kingdom In this study we tested the commonly held assumption that stress affects the appearance of skin through the premature occurrence of clinical features of skin ageing (lines, wrinkles and dark under-eye circles). We recruited 84 healthy adult female volunteers (age range, 24 e 40 years; mean 30.7 4.7 years); each volunteer completed a comprehensive battery of psychological and sleep questionnaires (State-Trait Anxiety Inventory, Perceived Stress Score, Hospital Anxiety and Depression Scale, Pittsburgh sleep quality index [PSQI], Ford insomnia response to stress test [FIRST] and consensus sleep diary), clinical photography (VISIA-CR ) and provided a sample of whole blood for epigenetic analysis. Image analysis of clinical photographs allowed the objective quantification of wrinkles around the eye (eye bag and crow’s feet), the nasolabial fold and deep mid-forehead frown lines. Initial analysis correlated perceived stress with worse sleep patterns; those volunteers who perceived stress more keenly had significantly disturbed sleep as measured by PSQI and FIRST with reduced sleep efficiency (r1⁄4-0.22; P<0.05) and increased wakefulness after sleep onset (r1⁄40.32; P<0.01). In addition, sleep onset latency positively correlated with increased measures for nasolabial folds, frown lines and crow’s feet wrinkles (r1⁄4-0.47; p<0.01). We next stratified the volunteers on the basis of their stress perception response to identify extreme groups (high stress response, n1⁄412; low stress response, n1⁄412) for epigenetic analysis of whole blood. Using Infinium HumanMethylation450BeadChip, principal component analysis identified significant clustering of methylation signatures between the two groups, with 484 differentially methylated regions observed (P<0.001). Hence, how an individual perceives psychological stress appears to impact on physical signs of skin ageing although the precise mechanism remains unclear.

360. Genotype/Phenotype Correlation of Cellular Function and AAV-Mediated Gene Delivery to Treat Xeroderma Pigmentosum - Cockayne Syndrome (XP - CS)

Cockayne Syndrome (CS) is a rare, autosomal recessive, neurodegenerative disorder characterized by deficiencies which all contribute to an overall phenotype of premature aging. The underlying cause is a defect in genes involving DNA repair mechanisms. This includes CSA and CSB as well as several genes associated with Xeroderma Pigmentosum (XP). Specifically, Xeroderma Pigmentosum Group G (XPG) is a disorder with two distinct clinical presentations: photosensitivity alone (XP) and photosensitivity with neurodegeneration (XP-CS).XPG's role as an endonuclease in nucleotide excision repair following UV exposure is well-described and explains the photosensitivity phenotype, yet a separate function for XPG that explains the neurological/early aging deficit that occurs in some patients remains obscure. Other CS proteins known to be involved in nuclear DNA repair have been shown to also function as free radical scavengers in mitochondria. As growing bodies of data illustrate the importance of mitochondria in aging, neuronal cell development and maintenance, and tissue repair, we hypothesize that XPG is trafficked to mitochondria where it could play an important role in mitochondrial function via free radical regulation. Such a deficiency could explain the neurodegeneration and multi-system early aging phenotype observed in CS patients. Our preliminary data (expansion rates, oxygen consumption, ATP generation, free radical sensitivity, Metronidazole [a drug that causes liver failure in CS patients] sensitivity) support this hypothesis through demonstration of decreased viability and mitochondrial function in fibroblasts derived from XPG patients displaying the XP-CS phenotype but not in those from patients displaying the XP phenotype alone. Mitochondrial isolations are being performed to confirm the presence of XPG in these organelles, as well as to better evaluate mitochondrial function.These characterizations will be used as outcome measures to determine the efficacy of AAV mediated gene therapy for XP-CS. A functional CMV-XPG plasmid has been cloned, and is being packaged into Adeno-associated virus (AAV). Further analyses in XP and XP-CS patient induced pluripotent stem cells differentiated into neurons and an XPG mouse model which closely replicates the human phenotype will yield useful information regarding the dual roles of this protein and provide data to support translation of gene therapy for CS. If successful, this will be the first therapeutic measure to demonstrate correction of the debilitating effects of XP-CS.

Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome

Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

182. Characterization and AAV-Mediated Correction of Xeroderma Pigmentosum-Cockayne Syndrome (XP/CS)

Cockayne Syndrome (CS) is caused by mutations in several genes that encode proteins involved in DNA repair. These include traditional CS proteins (CSA/CSB), as well as several Xeroderma Pigmentosum (XP) proteins (including XPG). While traditional CS is characterized by neurodegeneration and other clinical manifestations that result in an overall phenotype of premature aging, the typical XP phenotype is photosensitivity and skin cancer susceptibility. However, XPG presents with one of two different phenotypes, depending on the individual mutations: photosensitivity alone (XP) or photosensitivity + CS (XP/CS). Although the more severe XP/CS phenotype correlates well with mutations that result in XPG truncation, a function for this protein that explains the neurological deficit has yet to be described. XPG is involved in nuclear DNA repair as part of a complex that includes CSA and CSB. As CSA and CSB have also been shown to play a role in mitochondrial DNA (mtDNA) repair, it is likely, though unproven, that XPG is also involved in mtDNA repair. Insufficient mtDNA repair in neurons would ultimately lead to mitochondrial dysfunction and cause progressive neurodegeneration. The objective of this project is two-fold: 1) to characterize the two different phenotypes observed in XPG patients through examination of patient cells representing the XP as well as the XP/CS phenotype and compare them to healthy and CSA cells in order to identify a novel role for XPG that would explain the neurological deficit that occurs when the protein is truncated, and 2) to develop adeno-associated virus (AAV) mediated gene therapy for XPG and CSA and thus, overexpress these genes in an attempt to further characterize their function in healthy and patient-derived human cells. Cellular and mitochondrial function, sensitivity to oxidative and UV-induced stress, and general cell health and morphology are all being evaluated in healthy, XPG, and CSA patient-derived fibroblasts. Preliminary studies demonstrate reduced ATP production through oxidative phosphorylation in fibroblasts from patients with an XP/CS or CS phenotype as compared to those patients displaying XP alone or healthy controls. Scratch tests revealed motility and expansion deficiencies in XP/CS and CS cells as compared to XP or healthy controls, suggesting a potential role for mitochondria in these processes. AAV-XPG and AAV-CSA constructs have been generated and are being tested for their potential to correct cellular dysfunction. We are reprogramming a subset of these cells into induced pluripotent stem cells that will then be differentiated into neurons for further mitochondrial analyses and treatment studies. These studies will determine whether XPG function correlates with mitochondrial function in an in vitro human model of disease and generate preclinical data to justify further investigations into gene therapy strategies for this devastating syndrome.

The role of Cockayne Syndrome Protein B in transcription regulation.

We investigated the question if CSB (Cockayne Syndrome complementation B) protein actively regulates gene transcription and how mutations in CSB gene affect that regulatory role.Here we describe how we processed and interpreted ChIP-seq data (deposited in Gene Expression Omnibus with accession number GSE50171) obtained during an investigation of that question, and how this analysis assisted in the generation of hypothesis that were subsequently validated using other types of experiment.

Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth

Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients.

The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure.

Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.

Cockayne Syndrome: Varied Requirement of Transcription-Coupled Nucleotide Excision Repair for the Removal of Three Structurally Different Adducts from Transcribed DNA

Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER) pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS), however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N 2-yl)-2-acetylaminofluorene adduct (dG(N 2)-AAF) constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is removed exclusively by the CSA- and CSB-dependent pathway. In contrast, contribution of the CS proteins to the removal of two other transcription-blocking DNA lesions – N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG(C8)-AAF) and cyclobutane thymine-thymine (TT) dimer – is only minor (TT dimer) or none (dG(C8)-AAF). The unique properties of dG(N 2)-AAF identify this adduct as a prototype for a new class of DNA lesions that escape the alternative global genome repair and could be critical for the CS pathogenesis.

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS.

Multiple interaction partners for Cockayne syndrome proteins: Implications for genome and transcriptome maintenance

Cockayne syndrome (CS) is characterized by progressive multisystem degeneration and is classified as a segmental premature aging syndrome. The majority of CS cases are caused by defects in the CS complementation group B (CSB) protein and the rest are mainly caused by defects in the CS complementation group A (CSA) protein. Cells from CS patients are sensitive to UV light and a number of other DNA damaging agents including various types of oxidative stress. The cells also display transcription deficiencies, abnormal apoptotic response to DNA damage, and DNA repair deficiencies. Herein we have critically reviewed the current knowledge about known protein interactions of the CS proteins. The review focuses on the participation of the CSB and CSA proteins in many different protein interactions and complexes, and how these interactions inform us about pathways that are defective in the disease.

From laboratory tests to functional characterisation of Cockayne syndrome

The significant progress made over the last few years on the pathogenesis of Cockayne syndrome (CS) greatly improved our knowledge on several aspects crucial for development and ageing, demonstrating that this disorder, even if rare, represents a valuable tool to clarify key aspects of human health. Primary cells from patients have been instrumental to elucidate the multiple roles of CS proteins and to approach the dissection of the complex interplay between repair and transcription that is central to the CS clinical phenotype. Here we discuss the results of the cellular assays applied for confirmation of the clinical diagnosis as well as the results of genetic and molecular studies in DNA repair defective patients. Furthermore, we provide a general overview of recent in vivo and in vitro studies indicating that both CSA and CSB proteins are involved in distinct aspects of the cellular responses to UV and oxidative stress, transcription and regulation of gene expression, chromatin remodelling, redox balance and cellular bioenergetics. In light of the literature data, we will finally discuss how inactivation of specific functional roles of CS proteins may differentially affect the phenotype, thus explaining the wide range in type and severity of symptoms reported in CS patients.

Mitochondrial CSA and CSB: Protein interactions and protection from ageing associated DNA mutations

Cockayne syndrome (CS) is a rare autosomal recessive disease with progeroid symptoms, which is caused mainly by mutations in the CS genes CSA and CSB. Although the relevance of mitochondria in the aging process is known for several decades, research focused primarily on the role of the CS proteins in the nucleus. Recently, however, mitochondrial contribution to aging-associated symptoms of CS has been described. Inside mitochondria, CS proteins have roles, which partially differ from their nuclear functions. Up to now it is known that mitochondrial CS proteins are associated with base excision repair (BER) and transcription of mitochondrial DNA. However, it has been hypothesized that these are not the only functions of mitochondrial CS proteins as the occurrence of mutations like the common deletion are in need of separate explanations.

The role of CSA and CSB protein in the oxidative stress response

Cockayne syndrome (CS) is a rare hereditary disorder in which infants suffer severe developmental and neurological alterations and early death. Two genes encoding RNA polymerase II cofactors, CSA and CSB, are mutated in this syndrome. CSA and CSB proteins are known to be involved in the transcription-coupled DNA repair pathway but the sensitivity of mutant cells to a number of physical/chemical agents besides UV radiation, such as ionizing radiation, hydrogen peroxide and bioenergetic inhibitors indicate that these proteins play a pivotal role in additional pathways. In this review we will discuss the evidence that implicate CS proteins in the control of oxidative stress response with special emphasis on recent findings that show an altered redox balance and dysfunctional mitochondria in cells derived from patients. Working models of how these new functions might be key to developmental and neurological disease in CS will be discussed.