scholarly journals Immunofluorescence studies to dissect the impact of Cockayne syndrome A alterations on the protein interaction and cellular localization

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Amr Ghit
Keyword(s):  
Cellular Localization ◽  
Excision Repair ◽  
Growth Failure ◽  
Autosomal Recessive Disorder ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
Premature Aging ◽  
Neurological Dysfunction ◽  
C Terminus ◽  
The Impact

Abstract Background Cockayne syndrome (CS), which was discovered by Alfred Cockayne nearly 75 years ago, is a rare autosomal recessive disorder characterized by growth failure, neurological dysfunction, premature aging, and other clinical features including microcephaly, ophthalmologic abnormalities, dental caries, and cutaneous photosensitivity. These alterations are caused by mutations in the CSA or CSB genes, both of which are involved in transcription-coupled nucleotide excision repair (TC-NER), the sub-pathway of NER that rapidly removes UV-induced DNA lesions which block the progression of the transcription machinery in the transcribed strand of active genes. Several studies assumed that CSA and CSB genes can play additional roles outside TC-NER, due to the wide variations in type and severity of the CS phenotype and the lack of a clear relationship between genotype and phenotype. To address this issue, our lab generated isogenic cell lines expressing wild type as well as different versions of mutated CSA proteins, fused at the C-terminus with the Flag and HA epitope tags (CSAFlag-HA). In unpublished data, the identity of the CSA-interacting proteins was determined by mass spectrometry. Among which three subunits (namely, CCT3, CCT8, and TCP1) of the TRiC/CCT complex appeared as novel interactors. TRiC is a chaperonin involved in the folding of newly synthesized or unfolded proteins. The aim of this study is directed to investigate by immunofluorescence analysis the impact of the selected CSA mutations on the subcellular localization of the CSA protein itself as well as on its novel interactors CCT3, CCT8, and TCP1. Results We showed that specific CSA mutations impair the proper cellular localization of the protein, but have no impact on the cellular distribution of the TRiC subunits or CSA/TRiC co-localization. Conclusion We suggested that the activity of the TRiC complex does not rely on the functionality of CSA.

scholarly journals Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1671 ◽  
Author(s):  
Marios G. Krokidis ◽  
Mariarosaria D’Errico ◽  
Barbara Pascucci ◽  
Eleonora Parlanti ◽  
Annalisa Masi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
Premature Aging ◽  
Neurological Features ◽  
Oxygen Tensions ◽  
Base Excision

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

scholarly journals Base Excision Repair, a Pathway Regulated by Posttranslational Modifications

2016 ◽  
Vol 36 (10) ◽  
pp. 1426-1437 ◽  
Author(s):  
Rachel J. Carter ◽  
Jason L. Parsons
Keyword(s):  
Base Excision Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Cellular Localization ◽  
Excision Repair ◽  
Premature Aging ◽  
Base Excision ◽  
The Impact

Base excision repair (BER) is an essential DNA repair pathway involved in the maintenance of genome stability and thus in the prevention of human diseases, such as premature aging, neurodegenerative diseases, and cancer. Protein posttranslational modifications (PTMs), including acetylation, methylation, phosphorylation, SUMOylation, and ubiquitylation, have emerged as important contributors in controlling cellular BER protein levels, enzymatic activities, protein-protein interactions, and protein cellular localization. These PTMs therefore play key roles in regulating the BER pathway and are consequently crucial for coordinating an efficient cellular DNA damage response. In this review, we summarize the presently available data on characterized PTMs of key BER proteins, the functional consequences of these modifications at the protein level, and also the impact on BERin vitroandin vivo.

scholarly journals The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

1999 ◽  
Vol 10 (11) ◽  
pp. 3583-3594 ◽  
Author(s):  
Robert M. Brosh ◽  
Adayabalam S. Balajee ◽  
Rebecca R. Selzer ◽  
Morten Sunesen ◽  
Luca Proietti De Santis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Genetic Disorder ◽  
Cockayne Syndrome ◽  
Premature Aging ◽  
Atpase Domain ◽  
Acidic Region

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA andCSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, theCSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

scholarly journals Cockayne syndrome proteins CSA and CSB maintain mitochondrial homeostasis through NAD+ signaling

2020 ◽  
Author(s):  
Mustafa N. Okur ◽  
Evandro F. Fang ◽  
Elayne M. Fivenson ◽  
Vinod Tiwari ◽  
Deborah L. Croteau ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Excision Repair ◽  
Mitochondrial Dynamics ◽  
Accelerated Aging ◽  
Cockayne Syndrome ◽  
Premature Aging ◽  
Postmortem Brain ◽  
Average Life Expectancy ◽  
Postmortem Brain Tissue ◽  
Genes Encoding

AbstractBackgroundCockayne syndrome (CS) is a rare premature aging disease, most commonly caused by mutations of the genes encoding the CSA or CSB proteins. CS patients display cachectic dwarfism and severe neurological manifestations and have an average life expectancy of 12 years. The CS proteins are involved in transcription and DNA repair, with the latter including transcription-coupled nucleotide excision repair (TC-NER). However, there is also evidence for mitochondrial dysfunction in CS, which likely contributes to the severe premature aging phenotype of this disease. While damaged mitochondria and impaired mitophagy were characterized in mice with CSB deficiency, such changes in the CS nematodes and CS patients are not fully known.ResultsOur cross-species transcriptomic analysis in CS postmortem brain tissue, CS mouse and nematode models show that mitochondrial dysfunction is indeed a common feature in CS. Restoration of mitochondrial dysfunction through NAD+ supplementation significantly improved lifespan and healthspan in the CS nematodes, highlighting mitochondrial dysfunction as a major driver of the aging features of CS. In cerebellar samples from CS patients, we found molecular signatures of dysfunctional mitochondrial dynamics and impaired mitophagy/autophagy. In primary cells depleted for CSA or CSB, this dysfunction can be corrected with NAD+ supplementation.ConclusionsOur study provides support for the interconnection between major causative aging theories, DNA damage accumulation, mitochondrial dysfunction, and compromised mitophagy/autophagy. Together these three agents contribute to an accelerated aging program that can be averted by NAD+ supplementation.

scholarly journals CBIO-13. EPIGENETIC DEREGULATION OF NUCLEAR TRANSLOCATION - A NOVEL MECHANISM FOR TREATMENT RESISTANCE IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii18-ii18
Author(s):  
Thi Tham Nguyen ◽  
Premnath Rajakannu ◽  
Minh Diêu Thanh Pham ◽  
Leo Weman ◽  
Alexander Jucht ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Alkylating Agent ◽  
Nuclear Translocation ◽  
Excision Repair ◽  
Treatment Resistance ◽  
Dna Lesions ◽  
Aberrant Methylation ◽  
Knock Down ◽  
Endogenous Expression ◽  
A Genome

Abstract In a genome-wide DNA methylation analysis of glioblastoma we identified aberrant methylation of the HIV-1 Tat interactive protein 2 (HTATIP2) gene promotor. This was strongly correlated with downregulation of HTATIP2 gene expression, suggesting a potential tumor suppressor function in glioblastoma. HTATIP2 has been shown to inhibit nuclear translocation through interaction with β-importins. We hypothesize that deregulation of HTATIP2 expression inhibits nuclear entry of cancer-relevant proteins, thereby disturbing their specific function in the nucleus. We identified N-Methylpurine-DNA Glycosylase (MPG) as a potential cancer relevant candidate with the observation of nuclear and/or cytoplasmic localization in glioblastoma. MPG is a DNA repair protein that recognizes DNA lesions (N7-meG, N3-meA) and initiates base excision repair. We have shown that nuclear MPG expression contributes to resistance of glioblastoma to treatment with the alkylating agent temozolomide. Here we investigated the effect of HTATIP2 on cellular localization of MPG using (i) an inducible system (TET-ON) to express HTATIP2 in non-expressing glioblastoma cells, and (ii) HTATIP2-targeting siRNAs to knock down HTATIP2 in cells with endogenous expression. Results from confocal microscopy or Imagestream analyses showed a significant cytoplasmic retention of MPG in presence of HTATIP2, while knock-down of HTATIP2 resulted in nuclear MPG localization. Cytoplasmic retention of MPG in the presence of HTATIP2 was associated with a significant increase in γ-H2Ax signal after treatment with the alkylating agent: methyl methanesulfonate (MMS), suggesting increase in DNA damage. Mechanistically, we found that HTATIP2 co-localizes with importin β1, and excludes MPG localization. Furthermore, HTATIP2 displayed a similar effect on cytoplasmic retention of MPG as pharmacologic inhibition of Importin β 1. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and thereby contributing to treatment resistance.

scholarly journals Functional and clinical relevance of novel mutations in a large cohort of patients with Cockayne syndrome

2018 ◽  
Vol 55 (5) ◽  
pp. 329-343 ◽  
Author(s):  
Nadege Calmels ◽  
Elena Botta ◽  
Nan Jia ◽  
Heather Fawcett ◽  
Tiziana Nardo ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Cellular Response ◽  
Uv Light ◽  
Growth Failure ◽  
Cockayne Syndrome ◽  
Neurological Dysfunction ◽  
Compound Heterozygous ◽  
Founder Mutations ◽  
Multisystem Disorder ◽  
Cellular Analysis

BackgroundCockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS.Methods and resultsWe assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA.ConclusionBy identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS.

scholarly journals How (5′S) and (5′R) 5′,8-Cyclo-2′-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 725
Author(s):  
Karolina Boguszewska ◽  
Michał Szewczuk ◽  
Julia Kaźmierczak-Barańska ◽  
Bolesław T. Karwowski
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Genetic Material ◽  
Dna Lesions ◽  
The Other ◽  
Base Pairs ◽  
Ap Sites ◽  
Base Excision ◽  
The Impact ◽  
Ap Site

The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation’s impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5′,8-cyclo-2′-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus’ influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5′S) 5′,8-cyclo-2′-deoxyadenosine (ScdA), (5′R) 5′,8-cyclo-2′-deoxyadenosine (RcdA), (5′S) 5′,8-cyclo-2′-deoxyguanosine (ScdG) or (5′R) 5′,8-cyclo-2′-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5′-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3′-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5′-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

scholarly journals Cockayne Syndrome with ERCC8 Gene Mutation: A Case Report

2021 ◽  
Vol 44 (3) ◽  
pp. 181-183
Author(s):  
Kanij Fatema ◽  
Md Mizanur Rahman ◽  
Shaheen Akhter
Keyword(s):  
Case Report ◽  
Autosomal Recessive ◽  
Genetic Disorder ◽  
Growth Failure ◽  
Autosomal Recessive Disorder ◽  
Cockayne Syndrome ◽  
Global Developmental Delay ◽  
Hearing Disorders ◽  
Cognitive Delay ◽  
Intracerebral Calcification

Cockayne syndrome (CS) is a genetic disorder characterized by growth failure, microcephaly, cognitive delay, visual and hearing disorders. Patients usually present with dysmorphism and global delay. It is an autosomal recessive disorder, mutation of two genes ERCC8 and ERCC6 were observed. We report a 4 year old child who was diagnosed as a case of Cockayne syndrome, based on clinical, neuroimaging and genetic study findings. This case had growth failure, dysmorphism, optic atrophy, global developmental delay, intracerebral calcification and mutation of ERCC8 gene. Bangladesh J Child Health 2020; VOL 44 (3) :181-183

scholarly journals Two heterozygous mutations in the ERCC6 gene associated with Cockayne syndrome in a Chinese patient

2019 ◽  
Vol 48 (2) ◽  
pp. 030006051987799
Author(s):  
Qin Zhang ◽  
Minjuan Liu ◽  
Yinghua Liu ◽  
Hui Tang ◽  
Ting Wang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Excision Repair ◽  
Gene Mutations ◽  
Chinese Patient ◽  
Cockayne Syndrome ◽  
Premature Aging ◽  
Compound Heterozygous ◽  
Type I ◽  
Insertion And Deletion ◽  
Prenatal Diagnostic

Objective To confirm diagnosis and explore the genetic aetiology in a Chinese patient suspected to have Cockayne syndrome (CS). Methods The patient was clinically examined, and the patient and her biological parents underwent genetic analysis using whole exome sequencing (WES) and Sanger sequencing. The foetus of the patient’s mother underwent prenatal diagnostic Sanger sequencing using amniotic fluid obtained at 19 weeks’ gestation. Results Clinical examination of the patient showed developmental delay, progressive neurologic dysfunction and premature aging. Two compound, heterozygous ERCC excision repair 6, chromatin remodelling factor ( ERCC6) gene mutations were detected in the proband by WES and confirmed by Sanger sequencing, comprising a known paternal nonsense mutation (c.643G > T, p.E215X) and a novel maternal short insertion and deletion mutation (c.1614_c.1616delGACinsAAACGTCTT, p.K538_T539delinsKNVF). The patient was consequently diagnosed with CS type I. The foetus of the patient’s mother was found to carry only the maternally-derived c.1614_c.1616delGACinsAAACGTCTT variant. Conclusion This study emphasized the value of WES in clinical diagnosis, and enriched the known spectrum of ERCC6 gene mutations.

scholarly journals Retinal Degeneration and Ionizing Radiation Hypersensitivity in a Mouse Model for Cockayne Syndrome

2006 ◽  
Vol 27 (4) ◽  
pp. 1433-1441 ◽  
Author(s):  
Theo G. M. F. Gorgels ◽  
Ingrid van der Pluijm ◽  
Renata M. C. Brandt ◽  
George A. Garinis ◽  
Harry van Steeg ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Mouse Model ◽  
Photoreceptor Cells ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
Premature Aging ◽  
Oxidative Stress Marker ◽  
Marker Genes ◽  
Uv Sensitivity ◽  
Transcription Coupled Repair

ABSTRACT Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csb m/m mice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csb m/m retina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csb m/m mice (as well as Csa −/− mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csb m/m retina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging.

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