Project description:XPA is a central scaffold protein in nucleotide excision repair (NER) that interacts with and coordinates the assembly of repair complexes. Inactivating mutations in XPA causes Xeroderma Pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA with a mild manifestation of XP without skin cancer, but with neurological features including cerebellar ataxia. We show that the mutant XPA protein shows a severely weakened interaction with the TFIIH complex leading to its inefficient association with NER complexes and an inability to support the efficient association of the ERCC1-XPF endonuclease with repair complexes. Despite these defects, we find that patient-derived fibroblasts and reconstituted knockout cells carrying the H244R substitution show considerable levels of residual global genome repair (~40%), which is in intrinsic property of the mutated protein as revealed by in vitro experiments. In contrast, patient fibroblasts and engineered cells only expressing the mutant XPA protein were fully deficient in transcription-coupled repair. We report a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled sub-pathway of nucleotide excision repair, which is more closely associated with neurological features than to skin abnormalities.
Project description:We demonstrate that transcriptomic profiling of the NER mutant ercc-1 offers better understanding of the complex phenotypes of ercc-1 deficiency in C. elegans, as it does in mammalian models. There is a transcriptomic shift in ercc-1 mutants that suggests a stochastic impairment of growth and development, with a shift towards a higher proportion of males in the population. Extensive phenotypic analyses confirm that NER deficiency in C. elegans leads to severe developmental and growth defects and a reduced replicative lifespan, although post-mitotic lifespan is not affected. Results suggest that these defects are caused by an inability to cope with randomly occurring DNA damage, which may interfere with transcription and replication. The study investigates the developmental and aging phenotypes of different NER deficient C. elegans mutants (xpa-1, ercc-1, xpf-1 and xpg-1), where the transcriptomic profile of ercc-1 mutant is presented. We show that loss of NER function does not affect post-mitotic lifespan, but leads to impaired embryogenesis, germ cell and larval development and causes a reduced replicative lifespan. Phenotypes are most pronounced in ercc-1, xpf-1 and xpg-1 mutant animals. We provide evidence that this more pronounced phenotype is likely caused by the fact that these genes are involved in multiple repair pathways besides NER. Furthermore, transcriptional profiling of ercc-1 mutants confirms these observations, showing that growth and developmental pathways are underrepresented but that insulin signaling is not affected. Our analysis suggests that XPA-1, ERCC-1, XPF-1 and XPG-1 protect animals against replicative aging by preventing the accumulation of randomly acquired DNA damage. Eight mixed stage C. elegans samples were run on Affymetrix GeneChip C. elegans Genome Arrays. Four samples belong to ercc-1 mutant group and four to the wild-type, N2.
Project description:PxP-MS (Purification of x-linked Proteins coupled to Mass Spectrometry) was used to assess the role of CSB in DNA-protein crosslink repair. The CSB protein is a sensor that can detect stalled RNA polymerases at sites of DNA damage, thereby triggering transcription-coupled repair mechanisms. DPCs were induced in WT and CSB knock-out RPE1 cells using a pulse of formaldehyde. To identify crosslinked proteins that specifically require CSB for repair, DNA-protein crosslinks were isolated from cells using PxP either directly after formaldehyde exposure or following a chase in drug-free media and identified by mass spectrometry.
Project description:DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) riggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo.
Project description:Previous work with the classic T4 Endonuclease V digestion of irradiated Drosophila DNA followed by Southern hybridization led to the conclusion that Drosophila lacked transcription-coupled repair (TCR). This conclusion was reinforced by the Drosophila Genome Project which revealed that Drosophila lacked CSA, CSB, or UVSSA homologs, whose orthologs are present in eukaryotes that carry out TCR ranging from Arabidopsis to humans. A recently developed in vivo excision assay and the Excision Repair-sequencing (XR-seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single nucleotide resolution and strand-specific manner. Using these methods, we have discovered that Drosophila S2 cells carry out robust TCR comparable to that observed in mammalian cells. Our findings provide new insights into the mechanisms of TCR among various species.