Transcription-associated breaks in Xeroderma Pigmentosum group D cells from patients with combined features of Xeroderma Pigmentosum and Cockayne Syndrome

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded

Xeroderma pigmentosum: overview of pharmacology and novel therapeutic strategies for neurological symptoms

Xeroderma Pigmentosum (XP) encompasses a group of rare diseases characterised in most cases by nucleotide excision repair (NER) malfunction, resulting in an increased sensitivity to ultraviolet radiation in affected individuals. Approximately 25-30% of XP patients present with neurological symptoms, such as sensorineural deafness, mental deterioration, and ataxia. Although it is known that dysfunctional DNA repair is the primary pathogenesis in XP, growing evidence suggests that mitochondrial pathophysiology may also occur. This appears to be secondary to dysfunctional NER but may contribute to the neurodegenerative process in these patients. The available pharmacological treatments in XP mostly target the dermal manifestations of the disease. In the present review, we outline how current understanding of the pathophysiology of XP could be used to develop novel therapies to counteract the neurological symptoms. Moreover, the coexistence of cancer and neurodegeneration present in XP, lead us to focus on possible new avenues targeting mitochondrial pathophysiology

Dynamic Interaction of TTDA with TFIIH Is Stabilized by Nucleotide Excision Repair in Living Cells

Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER

Mutagenic and tumor suppressor functions of DNA polymerase iota in mammalian cells.

The Y family of DNA polymerases in higher eukaryotes contains at least four members which are implicated in potentially error-prone replication through unrepaired damage in the genome. These proteins are encoded by the REV1, POLH, POLI, and POLK genes. An inherited deficiency in one of these DNA polymerases (POL?) is the molecular defect in the cancer prone xeroderma pigmentosum (XP) variant syndrome, making POL? the most studied member of this family. However, there exist critical gaps in our knowledge on the function of the other known Y family members (POL I, POL K, and REV1). The goal of this proposal is to investigate the in vivo function of DNA POLI, and the hypothesis that DNA polymerase I acts as a mutagenic polymerase in translesion synthesis and as a tumor suppressor through a separate mechanism. To test this hypothesis, the mutagenic effects of chemical carcinogens which form structually different adducts will be examined in cells lacking POL? and/or POL I . To characterize the tumor suppressor function(s) of POL I, cell cycle progression will be monitored after UV in POL I null cells along with global gene expression. Finally, a novel mouse model will be used to determine the effect of pol? and/or polI deficiency on UV- and chemically-induced skin cancer

NUCLEOTIDE EXCISION REPAIR, CROSSLINK REPAIR AND TRANSCRIPTIONAL FUNCTION OF XPA IN HUMAN CELLS

Nucleotide excision repair (NER) in mammalian cells includes xeroderma pigmentosum group A protein (XPA) as a core factor. XPA and other NER proteins have been detected previously at some active promoters, and NER deficiency is reported to decrease activated transcription of selected genes. To determine the global extent of XPA influence on transcription, we analyzed the human transcriptome by RNA sequencing. We first confirmed that XPA is confined to the cell nucleus even in the absence of external DNA damage, in contrast to previous reports that XPA is normally resident in the cytoplasm and is imported following DNA damage. We then analyzed four genetically matched human cell line pairs deficient or proficient in XPA. At a false discovery rate of 0.05, 325 genes were common in all four pairs with a significant XPA-dependent directional change in gene expression. These genes were highly represented in pathways for the maintenance of mitochondria, metabolism and neurological system. Only 27 genes were regulated by more than 1.5 fold change. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism, and the corresponding proteins were lower in XPA-deficient cells. Transactivation by retinoic acid caused a modest enrichment of genes involved in transcription-related functions in XPA proficient cells. The results show that XPA status significantly influences a small subset of human genes that are important for mitochondrial and metabolic functions. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum (XP). An NER deficiency enhances sensitivity of mammalian cells to DNA interstrand crosslinks (ICL)-generating agents. I found that XPA is retained on damaged DNA following exposure to UVA-activated psoralen, and investigated repair of a triplex forming oligonucleotide (TFO)-directed psoralen ICL. A TFO-directed psoralen DNA ICL was constructed in closed-circular DNA. In NER proficient human cell extracts, incisions were detected on both strands of the damaged DNA 3’ to the psoralen ICL. Incision sites on the TFO bound strand were flanked by incision sites 40-42 nucleotides away from the ICL, with incisions 10-12 nucleotides away on the other strand

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders

Isolation of Xeroderma Pigmentosum - variant complementing factor from human 293 cells

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Sunlight damage to cellular DNA : focus on oxidatively generated lesions.

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells

Role of Pkc Delta in Uv Radiation Dna Damage Repair

DNA damage caused by ultraviolet radiation (UV), such as cyclobutane pyrimidine dimers (CPD), is repaired by the nucleotide excision repair (NER) pathway. When NER is defective, DNA damage is not repaired, leading to mutations and skin cancer. After DNA damage, the cell cycle is halted at various checkpoints to allow time for repair of the damage and maintain genomic integrity, however little is known about the coordination between NER DNA damage repair and cell cycle halting at checkpoints after DNA damage. Protein kinase C δ (PKCδ) plays major role in apoptosis and maintains the G2/M checkpoint in response to UV radiation, however PKCδ levels are low in squamous cell carcinomas. Since PKCδ is involved in UV-induced cell cycle checkpoints which are coupled to DNA damage repair, we hypothesized that PKCδ is also involved in repair of UV-induced DNA damage. Using immunofluorescence microscopy and flow cytometry, we found that murine embryonic fibroblasts (MEFs) lacking PKCδ are defective in their removal of UV-induced CPDs. In addition, PKCδ null MEFs had elevated mutagenesis frequency after UV exposure compared with wild type MEFs. We further wanted to investigate the mechanism behind the defective DNA damage repair. p53 was a prime suspect for this investigation because p53 is a major regulator of DNA damage repair and cycle checkpoints, and it has been reported to be regulated by PKCδ. We found that activating phosphorylation of p53 at serine 15 and total p53 levels were lower in PKCδ null MEFs after UV exposure compared to WT MEFs. Additionally, the UV induction of p53 target genes involved in cell cycle checkpoints (p21, GADD45a), but not NER genes (XPC, DDB2), was reduced in PKCδ null MEFs. Thus it can be speculated that the cell cycle checkpoint function of PKCδ may be a primary role for PKCδ in the UV DNA damage response. These findings suggest that loss of PKCδ expression would reduce repair of UV DNA damage, promote the accumulation of mutations, and potentially contribute to malignant transformation