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Xeroderma pigmentosum : comprendre la pigmentation en plus de l'initiation et de la progression du cancer de la peau / Xeroderma pigmentosum : understanding pigmentation in addition to the initiation and progression of skin cancerKasraian, Zeinab 03 July 2018 (has links)
Le xeroderma pigmentosum (XP) est une affection autosomique récessive avec déficience dans un système de réparation de l'ADN appelé réparation de l'excision des nucléotides (NER). Le XP comprend 7 groupes de complémentation, de XPA à XPG. Le xeroderma pigmentosum type C (XPC) est l'une des formes les plus courantes en Europe, aux Etats-Unis et au Japon. Les hétérozygotes ne sont pas affectés, alors que les homozygotes ont une sensibilité au soleil précoce et sévère qui endommage la peau et les yeux. La maladie commence au début de la vie avec la première exposition à la lumière du soleil. L'âge médian d'apparition est de 1 à 2 ans, la peau présentant rapidement les signes associés habituellement à des années d'exposition au soleil. Le terme XP fait référence au phénotype qui inclut la xérose et l'hyperpigmentation. Cependant, l'altération du phénotype pigmentaire de la peau associée aux effets du rayonnement UV, qui est parmi les premières manifestations cliniques dans la plupart des troubles NER, a été relativement négligée jusqu'à présent, sans étude systématique. En conséquence, l'objectif principal de cette thèse est d'étudier le mécanisme par lequel la déficience en protéine XPC affecte la pigmentation. Nos données cliniques ont révélé que les manifestations pigmentaires chez les patients XP-C apparaissent progressivement pour être manifestes vers l'âge de deux ans sous forme, dans les zones exposées au soleil, de nombreuses taches hypo- et hyper-pigmentées. De plus, le phénotype pigmentaire (c'est-à-dire la distribution des taches hypo- et hyper-pigmentées) des patients XP-C examiné sur une longue période de temps a révélé un profil relativement stable dans l'espace. L'analyse des zones hypo- et hyper-pigmentées de la peau chez les patients XP-C a montré que le nombre de mélanocytes et la quantité de mélanine sont significativement diminués dans la zone hypo pigmentée. L'analyse protéomique sur les taches hypo- et hyper-pigmentées a été réalisée et a permis d’identifier des voies de signalsation d’intérêt. Pour étudier en détail le mécanisme moléculaire soustendant l'effet de la déficience en XPC sur la pigmentation, nous avons utilisé la peau reconstruite humaine, qui reproduit de nombreuses caractéristiques morphologiques et moléculaires de la peau humaine. Les résultats montrent que l'extinction du gène XPC dans les mélanocytes en utilisant la technologie CRISPR/ Cas9 pourrait être utilisé comme un modèle pour reproduire la maladie en raison de la rareté des patients. / Xeroderma pigmentosum (XP) is an autosomal recessive disorder with deficiency in aDNA repair system called nucleotide excision repair (NER). XP comes into 7 complementationgroups, XPA-XPG. Xeroderma pigmentosum type C (XPC) is one of the morecommon forms in Europe, USA and Japan. Heterozygotes are unaffected, while homozygoteshave an early and severe sun sensitivity which damages the skin and eyes.The disease begins in early life with the first exposure to sunlight. The median age ofonset is 1–2 years of age, with skin rapidly exhibiting the signs associated with years ofsun exposure. The term XP refers to the phenotype that includes xerosis and hyperpigmentation.However, alteration in the skin pigmentary phenotype associated with theeffects of UV radiation which is among the first clinical manifestations in most of theNER disorders, has been neglected up to now and it has not studied in detail. Accordingly,the main objective of this thesis is to investigate the mechanism by which XPC deficiencyaffects pigmentation.Our clinical data revealed that pigmentary manifestations in XP-C patients often appearprogressively and are characteristic by two years of age as an abnormal increasednumber of lentigines in sun-exposed areas with the typical appearance of hypo- andhyperpigmentation spots. Moreover, pigmentary phenotype (i.e. distribution of hypoandhyper-pigmented spots) of XP-C patients examined over a long period of time revealeda relatively spatially stable profile. Analysis of the hypo- and hyper-pigmentedareas of the skin isolated from XP-C patients showed that the number of melanocytesand melanin quantity are significantly decreased in the hypo-pigmented area. Proteomicanalysis on hypo- and hyper-pigmented spots identified some molecular targets thatwere expressed differently in these spots. To investigate in detail the molecular mechanismunderlying the effect of XPC deficiency on pigmentation, human reconstructedskin, which mimics many morphological and molecular characteristics of normal humanskin was used for modeling XPC. Our results showed that silencing of XPC inmelanocyte using CRISPR/ Cas9 technology could be used as a useful model to counteractthe scarcity of patient samples.
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Developmental Characteristics of Mice Lacking the DNA Excision Repair Gene XPGSUN, Xue-Zhi, HARADA, Yoshi-Nobu, GUI, Chun, ZHANG, Rui, TAKAHASHI, Sentaro, Fukui, Yoshihiro, MURATA, Yoshiharu 12 1900 (has links)
国立情報学研究所で電子化したコンテンツを使用している。
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Estudo de polimorfismos de genes de reparo do DNA em lesão de fita simples e sua associação com aspectos clínicos e laboratoriais de portadores de síndrome mielodisplásica / Study of dna repair gene polymorphisms in single-stranded lesion and its association with clinical and laboratory aspects of patients with myelodysplastic syndromeSantiago, Sabrina Pinheiro 16 December 2015 (has links)
SANTIAGO,S.P. Estudo de polimorfismos de genes de reparo do DNA em lesão de fita simples e sua associação com aspectos clínicos e laboratoriais de portadores de síndrome mielodisplásica, 2015. 76 f. Dissertação (Mestrado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza,2015 / Submitted by Ivone Sousa (ppgcm.ufc@gmail.com) on 2017-08-02T13:51:15Z
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Previous issue date: 2015-12-16 / Myelodysplastic Syndrome (MDS) is a group of clonal diseases of hematopoietic progenitor cells, characterized by peripheral cytopenia (s), dysplasia of one or more myeloid cell lines and increased risk of developing acute myeloid leukemia. The pathogenesis of SMD involves DNA damage in hematopoietic stem cells likely to be affected by single-stranded DNA damage and the Nucleotide Excision Repair (NER) as one of the main mechanisms to ensure the genomic stability of cells -trunk. This case-control study of candidate genes proposed to evaluate the association of the polymorphisms XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067 with the clinical variables of patients with Myelodysplastic Syndrome. This analysis used real-time quantitative PCR as a technique for genotyping between bone marrow samples from 95 MDS patients from the Walter Cantídio University Hospital and peripheral blood samples from 94 healthy elderly volunteers. For the polymorphism XPC rs2228000, we obtained, in relation to hemoglobin, in the genotypic distribution, that the CT genotype is associated with a higher odds ratio of hemoglobin levels below 8.0 g / dL (p = 0.010, OR 3.385, CI 1.343- 8,529); (P = 0.027, OR 2.75, CI 1.125-6.722) and in the dominant heterozygosis model (p = 0.013). In the present study, the genotype CC was associated with a higher odds ratio of Hb ≥ 8g / dL in the dominance model , OR 3.333, CI: 1.293-8.591); In relation to neutrophil counts we have that the mutant TT genotype has a higher odds ratio of neutrophil counts below 800 / mm3 (p = 0.017, OR 8,750, CI 1,470-52,098), that the CC genotype is associated with the highest ratio (P = 0.032, OR 2,833, CI 1,096-7,327) and in the homozygous model (p = 0.013, OR 17,500, CI 1,828-167,558) and that the combination CC + CT Of the recessive model was also associated with a higher odds ratio of neutrophils ≥ 800 / mm³ (p = 0.025, OR 12,500, CI 1,381-113,177). We also found that for genotype XPD rs1799793 the GG genotype is associated with a lower odds ratio of SMD in the dominance model (p = 0.009, OR 0.445, CI 0.242-0.816) and in the dominant heterozygosis model (p = 0.010, OR 0.440 , IC 0.235-0.825), and the same genotype seems to have a lower odds ratio for bone marrow ring sideroblast counts ≥ 15% in three different models: genotype distribution (p = 0.027, OR 0.980, CI 0.013-0.768) , Dominance (p = 0.027, OR, 4,643, CI 1,195-18,034) and dominant heterozygosity (p = 0.022, OR 5,000, CI 1,265-19,762). We did not find a significant association between the XPF polymorphisms rs1800067 and XPA rs1800975 and the clinical variables for patients with MDS. This study suggests that polymorphisms of DNA repair genes by nucleotide excision are associated with clinical features of patients with myelodysplastic syndrome. / A Síndrome Mielodisplásica (SMD) é um grupo de doenças clonais das células progenitoras hematopoéticas, caracterizadas por citopenia(s) periférica(s), displasia de uma ou mais linhagens celulares mielóides e aumento do risco de desenvolvimento de leucemia mielóide aguda. A patogênese da SMD envolve danos no DNA nas células-tronco hematopoéticas acometidos provavelmente pelos danos de fita simples no DNA tendo o processo de reparo por excisão de nucleotídeos (Nucleotide excision repair – NER) como um dos principais mecanismos para garantir a estabilidade genômica das células-tronco. Este estudo de caso-controle de genes candidatos propôs avaliar a associação dos polimorfismos XPA rs1800975, XPC rs2228000, XPD rs1799793 e XPF rs1800067 com as variáveis clínicas de pacientes com Síndrome Mielodisplásica. Esta análise utilizou PCR quantitativo em tempo real como técnica de genotipagem entre amostras de medula óssea de 95 pacientes com SMD, oriundos do Hospital Universitário Walter Cantídio, e amostras de sangue periférico de 94 idosos voluntários sadios. Para o polimorfismo XPC rs2228000 obtivemos, em relação a hemoglobina, na distribuição genotípica, que o genótipo CT é associado a maior razão de chances de apresentar níveis de hemoglobina abaixo de 8,0g/dL (p= 0,010, OR 3,385, IC 1,343-8,529); por sua vez, o genótipo CC associa-se a maior razão de chances de Hb ≥ 8g/dL no modelo de dominância (p= 0,027, OR 2,75, IC 1,125-6,722) e no modelo de heterozigose dominante (p= 0,013, OR 3,333, IC: 1,293-8,591); em relação a contagem de neutrófilos temos que o genótipo mutante TT tem maior razão de chances de contagem de neutrófilos abaixo de 800/mm3 (p= 0,017, OR 8,750, IC 1,470-52,098), que o genótipo CC associa-se a maior razão de chances de neutrófilos ≥ 800/mm3 no modelo de dominância (p= 0,032, OR 2,833, IC 1,096-7,327) e no modelo de homozigose (p= 0,013, OR 17,500, IC 1,828-167,558) e que a combinação CC+CT do modelo de recessividade também associou-se com maior razão de chances de neutrófilos ≥ 800/mm³ (p= 0,025, OR 12,500, IC 1,381-113,177). Constatamos ainda que para o polimorfismo XPD rs1799793 o genótipo GG se associa a menor razão de chances de SMD no modelo de dominância (p= 0,009, OR 0,445, IC 0,242-0,816) e no modelo de heterozigose dominante (p= 0,010, OR 0,440, IC 0,235-0,825), e o mesmo genótipo parece ter menor razão de chances de contagem de sideroblastos em anel em medula óssea ≥ 15% em três modelos distintos: de distribuição genotípica (p= 0,027, OR 0,980, IC 0,013-0,768), de dominância (p= 0,027, OR 4,643, IC 1,195-18,034) e de heterozigose dominante (p= 0,022, OR 5,000, IC 1,265-19,762). Não obtivemos associação significante entre os polimorfismos XPF rs1800067 e XPA rs1800975 e as variáveis clínicas para os pacientes com SMD. Este estudo sugere que os polimorfismos de genes de reparo do DNA por excisão de nucleotídeos são associados a características clínicas de pacientes com Síndrome Mielodisplásica.
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Role of NER factors in transcription / Rôle des facteurs NER dans la transcriptionCostanzo, Federico 17 November 2017 (has links)
Les mutations dans les gènes codant pour les facteurs NER donnent lieu à des maladies autosomiques récessives telles que Xeroderma pigmentosum (XP), le syndrome de Cockayne (CS) et la trichothiodystrophie (TTD). Les phénotypes associés à ces syndromes génétiques se caractérisent par une sensibilité extrême à la lumière UV, avec prédisposition accrue à certains cancers (pour XP et XP / CS combiné, principalement), ainsi qu’un retard mental et des signes de progeria (pour CS et XP / CS combiné). Si on peut admettre une corrélation entre réparation de l'ADN endommagé et sensibilité aux UV / cancer, celle avec les symptômes neurologiques/progéroïdes est encore sujet à débat. Une explication pourrait provenir du rôle des facteurs NER dans la régulation de la transcription. Nous proposons une vue d’ensemble des roles de XPG et XPC dans la régulation de la transcription en absence des stress exogènes et comment CSA et CSB orchestrent l’arret de la transcription après une attaque génotoxique. XPC était capable d’interagir stablement avec la methyltransferase NSD3. Des mutations dans XPC altèrent le transcriptôme et la distribution des H3K36me3. Les mutations dans XPG dérégulent l’expression génique et XPG est capable d’etre recruté sur l’ensemble du genôme avec TFIIH. CSA et CSB faisant partie de la machinerie ubiquitin/proteasôme, régulent le recrutement de facteurs fixant l’ADN et contrôlant le programme transcriptionnel après irradiation aux UV. Nos donnés mettent en évidence le rôle des facteurs NER dans la transcription et leur défaut d’action provoque les maladies XP et XP/CS. En plus, nos données fournissent des explications sur le méchanisme d’arrêt de la transcription après un stress genotoxique et pose la question de l’origine du phenotype CS. / Mutations in genes coding for NER factors give rise to autosomal recessive diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). The phenotypes associated with these genetic syndromes spans from extreme sensitivity to UV light, with increased predisposition to cancer (for XP and combined XP/CS, mostly), mental retardation and progeria (for CS and combined XP/CS). Whether the correlation between defective DNA repair reactions and UV-sensitivity/cancer may be more intuitive, a link with neurological/progeroid symptoms is still a matter of debate. As a possible explanation, it has been proposed a connection between NER and transcription regulation. We propose additional insights on XPG and XPC roles in transcription regulation in absence of exogenous stress and how CSA and CSB orchestrate transcription arrest due to genotoxic attack. XPC was able to stably interact with NSD3 methyltransferase. Mutations in XPC also disturbed the transcriptome and the H3K36me3 distribution. Mutations in XPG deregulate gene expression and XPG is able to be recruited genome wide together with TFIIH. CSA and CSB can, as part of the ubiquitin/proteasome machinery, regulate the recruitment timing of DNA binding factors and control transcriptional program after UV irradiation. Hence, our data shed more light in NER factors role in transcription and their defective action as a cause of XP and XP/CS disorders. Additionally, our data provide explanations on the mechanism of transcription arrest following genotoxic stress and pose questions about the origins of CS phenotype.
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Construction of a Herpes Simplex Virus Type 1 (HSV-1) Expression Vector Containing the Bacteriophage T4 Den V Gene: Effect of this Gene on UV-Survival of HSV-1 in Normal and Zeroderma Pigmentosum Fibroblasts / Construction of an HSV-1 Recombinant Expressing the Bacteriophage T4 Den V GeneTang, Katherine 09 1900 (has links)
In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)
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Reactivation of UV-Irradiated Herpes Simplex Virus Type 2 in Cockayne's Syndrome and Xeroderma Pigmentosum Cells / Reactivation of UV-Irradiated Herpes Simplex Virus Type 2 in Human CellsRyan, David 04 1900 (has links)
Host cell reactivation (HCR) of UV-irradiated (UV'd) herpes simplex virus type 2 (HSV-2), capacity of UV'd cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV'd HSV-2 were examined in human fibroblasts. The cells were derived from four Cockayne's Syndrome (CS) patients, 5 xeroderma pigmentosum (XP) patients and 5 normal patients.
Survival curves for HCR of HSV-2 plaque formation showed 2- components. HCR was not significantly different in the CS
strains and an XP variant strain compared to normal, whereas all excision deficient strains showed a significant reduction
in HCR. The o37 values for the delayed capacity curves were in the range 8.6-12.4 J/m2 for the normal strains, 3.1-5.1
J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.40-1.98 J/m2 for the XP excision deficient strains
examined. UVER was also examined for HSV-2 UV-irradiated to survival levels of 10-2 and 10-3 in unirradiated cells. Maximum
delayed UVER was observed in normal strains at a UV dose of 15 J/m2 to the virus. Maximum UVER in CS cells was detected at a UV dose of 5 J/m2 to the cells, in XP excision deficient cells maximum UVER occurred at doses ranging from 0.5-2.5 J/m2 to the cells, and in XP variant maximum UVER occurred at 10 J/m2 to the cells. In all cell strains the level of UVER increased with increasing UV dose to the virus. Results are discussed in terms of the repair defects of CS and XP cells and their relationship to possible viral repair functions. In addition, the possible existence of an inducible DNA repair response is discussed in terms of the results of this study. / Thesis / Master of Science (MSc)
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Biochemical characterization of the TFIIH translocase XPB from \(Chaetomium\) \(thermophilum\) / Biochemische Charakterisierung der TFIIH Translokase XPB aus \(Chaetomium\) \(thermophilum\)Kappenberger, Jeannette Sarah January 2024 (has links) (PDF)
DNA repair and gene expression are two major cellular processes that are fundamental for the maintenance of biological life. Both processes require the enzymatic activity of the super family 2 helicase XBP, which is an integral subunit of the general transcription factor TFIIH. During transcription initiation, XPB catalyzes the initial melting of promoter DNA enabling RNA polymerase II to engage with the coding DNA strand and start gene transcription. In nucleotide excision repair, XPB acts in concert with the other TFIIH helicase XPD causing strand separation around a lesion site. Mutations within the genes encoding XPB or other TFIIH subunits are associated with different cancer types as well as with the autosomal recessive disorders Xeroderma Pigmentosum and trichothiodystrophy and rarely combined features of Xeroderma Pigmentosum and Cockayne syndrome.
In the last few years, great progress has been made towards unraveling the structure of TFIIH and its individual subunits including XPB. These structural insights tremendously improved our understandings with respect to the molecular interactions within this intriguing protein complex. However, the underlying regulation mechanisms that functionally control XPB during transcription and repair remained largely elusive. We thus executed the biochemical characterization of this protein to investigate the functional network that regulates XPB within the scaffold of TFIIH. Due to their enhanced stability compared to the human proteins, we utilized the proteins that originate from the thermophilic fungus Chaetomium thermophilum for this purpose as a model organism for eukaryotic TFIIH.
The present work provides novel insights into the enzymatic function and regulation of XPB. We could show that both, DNA and the TFIIH subunit p52 stimulate XPB’s ATPase activity and that the p52-mediated activity is further boosted by p8, another subunit within TFIIH. Surprisingly, DNA can activate XPB’s ATPase activity to a greater extent than its TFIIH interaction partners p52/p8, but when both, i.e. p52/p8 and DNA are present at the same time, p52 dominates the activation and the enzymatic speed is maintained at the level observed through the sole activation of p52/p8. We thus defined p52 as the master regulator of XPB that simultaneously activates and represses XPB’s enzymatic activity. Based on a correlative mutagenesis study of the main interface between p52 and XPB that was set into context with recent structural data, a model for the p52-mediated activation and speed limitation of XPB’s ATPase was proposed. The research on XPB’s ATPase was expanded with the investigation of the inhibition mechanism of XPB’s ATPase via the natural compound Triptolide. Furthermore, we investigated XPB’s DNA translocase function and could observe that XPB can only perform its translocase movement when it is fully incorporated into core TFIIH and this translocase movement is further enhanced by the nucleotide excision repair factor XPA. Fluorescence polarization measurements with nucleotide analogues revealed that XPB displays the highest affinity towards DNA in the ADP + Pi bound state and its binding is weakened when ADP is bound or the nucleotide is dissociated from the enzyme, suggesting a movement on the DNA during the distinct states of the ATPase cycle. Finally, the well-known and highly conserved RED motif was found to be the crucial element in XPB to enable this translocase movement. Combined, the data presented in this work provide novel insights into the intricate regulation network that controls XPB’s enzymatic activity within TFIIH and furthermore show that XPB’s enzymatic activity is tightly controlled by various factors. / DNA Reparatur und Genexpression sind zwei fundamentale zelluläre Prozesse die unabdingbar für die Aufrechterhaltung des biologischen Lebens sind. Beide Prozesse benötigen die Enzymaktivität der Superfamilie 2 Helikase XPB, welche eine Untereinheit des Transkriptionsfaktors TFIIH darstellt. Während der Transkriptions-Initiation katalysiert XPB das initiale Aufschmelzen der Promoter-DNA und befähigt dadurch die
RNA-Polymerase II dazu an den codierenden DNA Strang zu binden und die Genexpression zu starten. In der Nukleotid-Exzisions-Reparatur agiert XPB zusammen mit der zweiten TFIIH Helikase, XPD, und bewirkt die Öffnung des DNA Stranges an der Stelle des DNA-Schadens. Mutationen des XPB-Gens oder der Gene der anderen TFIIH
Untereinheiten sind mit verschiedenen Krebsarten, sowie den autosomal rezessiv vererbten Krankheiten Xeroderma Pigmentosum und Trichothiodystrophie assoziiert. In seltenen Fällen kann eine kombinierte Form von Xeroderma Pigmentosum und Cockayne Syndrom auftreten.
In den letzten Jahren wurde mittels der Cryo-EM die Strukturaufklärung von TFIIH und seinen Untereinheiten einschließlich XPB signifikant vorangebracht. Diese neuen
strukturellen Einsichten haben unser Verständnis über den molekularen Aufbau des TFIIH Komplexes entscheidend verbessert. Jedoch sind die Regulationsmechanismen, die XPB auf funktionaler Ebene kontrollieren, noch größtenteils unbekannt. Um das funktionelle Netzwerk, das XPB innerhalb von TFIIH reguliert, zu erforschen, haben wir die biochemische Charakterisierung von XPB verfolgt. Aufgrund ihrer erhöhten Stabilität gegenüber den humanen Proteinen wurden für diese Analyse die Proteine des
thermophilen Pilzes Chaetomium thermophilum als Modellorganismus für TFIIH verwendet.
Die vorgelegte Arbeit liefert neue Erkenntnisse über die enzymatische Funktion und Regulation von XPB. Wir konnten zeigen, dass sowohl DNA, als auch die TFIIH
Untereinheit p52 die ATPase Aktivität von XPB stimulieren und dass die p52-vermittelte Aktivierung durch p8, eine weitere Untereinheit von TFIIH, noch weiter verstärkt wird. In Gegenwart von DNA beobachtet man jedoch die höchste ATPase Aktivität. Wenn beide Aktivatoren, also p52/p8 und DNA, gleichzeitig anwesend waren, dominierte die niedrige p52-vermittelte Aktivierung gegenüber der DNA-vermittelten Aktivierung. Das p52-Protein agiert also als Aktivator und Deaktivator indem es die enzymatische Aktivität des XPB-Proteins gleichzeitig aktiviert und hemmt und kann damit folglich als Hauptregulator von XPB bezeichnet werden. Basierend auf einer korrelativen Mutagenese-Analyse der Interaktionsfläche zwischen p52 und XPB sowie auf den aktuellsten Strukturdaten, wurde ein Modell für die p52-vermittelte Aktivierung und Geschwindigkeitsregulierung von XPBs ATPase generiert. Des Weiteren wurde der Einfluss des Naturproduktes Triptolid auf die Hemmung der enzymatischen Aktivität des XPB Proteins untersucht. Darüber hinaus haben wir die doppelsträngige DNA-Translokase-Aktivität von XPB
analysiert und konnten feststellen, dass die Translokation nur erfolgen kann, wenn XPB vollständig in den Kern-TFIIH-Komplex integriert ist. Der Nukleotid-Exzisions-Reparatur-
Faktor XPA stimulierte diese Translokase-Aktivität zusätzlich. Fluoreszenz-Polarisations-Messungen mit Nukleotid-Analoga zeigten, dass XPB die höchste Affinität
für DNA im ADP + Pi gebundenen Zustand aufweist und dass diese Bindung gelockert wird, wenn ADP gebunden oder das Nukleotid dissoziiert ist. Dies deutet auf einen
Bewegungsmechanismus auf der DNA während der verschiedenen Stadien des ATPase-Zyklus hin. Abschließend konnten wir zeigen, dass das hochkonservierte RED-Motiv eine entscheidende Rolle für die Translokase Bewegung des XPB-Proteins einnimmt. Zusammenfassend präsentiert diese Arbeit neue Erkenntnisse, die unser Verständnis des Regulierungsnetzwerkes, das die enzymatische Aktivität von XPB innerhalb von TFIIH steuert, entscheidend vorangebracht haben.
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Développement d’un modèle de correction génétique du xeroderma pigmentosum par recombinaison homologue ciblée par des endonucléases ingéniérées / Model of gene correction of xeroderma pigmentosum mediated by engineered endonuclease-induced homologous recombinationDupuy, Aurélie 20 December 2012 (has links)
Le xeroderma pigmentosum (XP) est une maladie génétique rare caractérisée par une hypersensibilité aux ultraviolets (UV) et une forte incidence des tumeurs cutanées. Les cellules des patients XP sont incapables d’éliminer les lésions induites dans l’ADN par les UV en raison d’un dysfonctionnement du mécanisme de réparation par excision de nucléotides (NER). Plusieurs groupes de complémentation ont été identifiés dans le syndrome XP, parmi lesquels le groupe XP-C représente la majorité des patients à travers le monde.Au cours de mon travail de thèse, j’ai développé un modèle de correction ciblée par recombinaison homologue (RH) d’une délétion de deux nucléotides au niveau de l’exon 9 du gène XPC aboutissant à l’apparition prématurée d’un codon stop. Afin de stimuler la RH, deux types de nucléases ingéniérées sont utilisées : les méganucléases et les TALENs. J’ai observé que la méthylation de la séquence ciblée pouvait affecter l’activité de celles-ci et donc l’efficacité du ciblage de gène. Cependant, deux approches ont été développées pour résoudre ce problème : l’utilisation d’un agent déméthylant (5-aza-2’-désoxycytidine (5azadC)) ou la création d’une endonucléase insensible à la méthylation. L’utilisation des méganucléases en combinaison avec la 5azadC a permis de stimuler la fréquence de coupure de presque 20 fois dans des fibroblastes XPC et la TALEN modifiée permet une augmentation de 40 fois. Avec ces deux stratégies j’ai obtenu des événements de correction génétique par introduction d’une matrice de réparation dans le locus ciblé avec une fréquence proche de 3%. La caractérisation des clones corrigés avec la TALEN XPC montre la correction génomique des deux nucléotides dans l’exon 9, une restauration de l’expression de la protéine XPC et une résistance cellulaire après irradiation UV traduisant le rétablissement des fonctions de la NER. Cette étude représente la première preuve de correction génétique de cellules déficientes en protéine XPC en utilisant une approche ciblée. / Xeroderma pigmentosum (XP) is a rare inherited genetic disorder characterized by an UV hypersensitivity and a severe predisposition to skin cancers. Cells from XP patients are deficient in nucleotide excision repair (NER) of UV‐induced DNA lesions. Several complementation groups have been identified in the XP syndrome and the XP-C group represents the majority of XP patients around the world. During my PhD work, I developed a model of targeted correction by homologous recombination (HR) in order to correct a deletion of two nucleotides in the ninth exon in XPC gene leading to a premature stop codon. To stimulate HR, I used two types of engineered endonucleases : meganucleases and TALEN. I observed that the target methylation status could affect the endonuclease activities and therefore XPC gene correction. Nervertheless, I developed two approaches to overcome this methylation sensitivity : use of a demethylating agent (5-aza-2-deoxycytidine (5azadC)) or a specific engineering of TALEN. Using 5azadC with meganuclease allowed to stimulate the cutting frequency by nearly 20 fold in XPC fibroblasts and the engineered TALEN allowed a 40 fold-increase in frequency. With both strategies I obtained genetic correction events by repair matrix introduction in the targeted locus with a near 3% frequency. The characterization of corrected clones with the XPC TALEN shows genomic correction in the ninth exon, a restoration of the XPC protein expression and cell survival following UV exposure, thus demonstrating fully recovered normal repair activity by NER. This study represents the first evidence of genetic correction of XPC-deficient cells by a targeted approach.
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Efeitos da luz UVA em células de pacientes com Xeroderma Pigmentosum Variante / Effects of UVA light on Xeroderma Pigmentosum Variant patient cellsMoreno, Natalia Cestari 11 October 2017 (has links)
Mais de 95% de luz ultravioleta (UV) que atinge a superfície da Terra corresponde ao comprimento de onda da luz UVA (315-400 nm). A luz UVA induz no DNA danos pela absorção direta e indireta dos fótons, bem como por intermédio de cromóforos. Pacientes com Xeroderma Pigmentosum Variante (XP-V) possuem mutações na DNA polimerase η (pol eta), que promove a síntese translesão dos danos induzidos pela luz solar. Na ausência dessa polimerase há aumento da mutagênese, provável causa de câncer de pele em pacientes XP-V. O objetivo deste trabalho foi caracterizar os mecanismos de indução de danos no genoma e mutagênese por luz UVA, em células derivadas desses pacientes. Os resultados indicam que a exposição à luz UVA resultou em maior sensibilidade de células XP-V comparadas à linhagem controle. Os níveis de fosforilação da histona H2AX (gerando γH2AX - indicador de indução de danos no genoma) e detecção de danos diretos e indiretos no DNA apresentou um aumento significativo em células XP-V irradiadas com luz UVA. Curiosamente, na ausência de pol eta houve uma redução na capacidade de remoção das lesões formadas. Além disso, a irradiação com luz UVA causou forte bloqueio de replicação do DNA e parada do ciclo celular em fase S em células XP-V, desencadeando importantes respostas mediadas por ATR/Chk1. Surpreendentemente, o antioxidante N-acetilcisteína (NAC) resultou em diminuição da sensibilidade celular, dos níveis de γH2AX, da parada de forquilha de replicação e do ciclo celular, reduziu os efeitos citotóxicos da inibição de ATR, melhorou o reparo de lesões no DNA e preveniu a carbonilação de proteínas em células XP-V irradiadas com luz UVA. Investigamos também a indução de mutagênese nas células irradiadas com luz UVA, através de sequenciamento de exoma de clones celulares. Os dados indicaram um aumento significativo da mutagênese em células XP-V irradiadas comparadas a células controle, e pela avaliação dos tipos de mutações encontradas verificamos uma frequência bastante alta de transições C>T, provavelmente como consequência de replicação errônea de dímeros de pirimidina. Entretanto, também identificamos indução significativa de transversões C>A, provavelmente devido a lesões oxidadas no genoma. Curiosamente, ao compararmos células XP-V com células controle detectamos um aumento desse último tipo de mutação na ausência de pol eta, provavelmente devido a lesões endógenas, produzidas pela oxidação do DNA. Além disso, análises in silico mostraram que células XP-V irradiadas com UVA apresentaram assinatura mutacional similar ao observado para tumores de pele. Os dados claramente indicam que células XP-V são sensíveis a irradiação com UVA e que os danos promovidos no DNA, incluindo aqueles causados por estresse oxidativo, desencadeiam respostas celulares e mutagênese nesses pacientes. Assim, além de apontar que UVA pode gerar efeitos deletérios na pele de pacientes XP-V, nossos dados também contribuem para a compreensão de como esses comprimentos de onda podem atuar em células humanas em geral / More than 95% of ultraviolet-light (UV) that reaches the Earth surface corresponds to UVA wavelengths (315-400 nm). UVA-light induces DNA damage through direct and indirect absorption of photons, as well as, intermediated by chromophores and by oxidation mechanisms. Xeroderma Pigmentosum Variant (XP-V) patients are defective in DNA polymerase η (pol eta) that performs translesion synthesis of sunlight induced DNA damage. Absence of pol eta results in increased mutagenesis, which is probably responsible for high frequency of skin cancer in XP-V patients. The goal of this work was to characterize the mechanisms of UVA-induced genome DNA damage and mutagenesis in cells derived from these patients. The results indicate that UVA irradiation increased cell death of XP-V compared to control cell line. The phosphorylation of the histone H2AX (generating γH2AX, an indicator of genotoxic stress) and DNA damage was highly increased in UVA irradiated XP-V cells. Curiously, however, in the absence of pol eta, there was a reduction in the capacity of cells to remove DNA damage from genome. Moreover, UVA irradiation triggered strong DNA synthesis blockage and cell cycle arrest in S phase, resulting in important responses mediated by the ATR/Chk1 pathway in XP-V cells. Interestingly, the antioxidant N-acetyl cysteine (NAC) resulted in decreased cell sensitivity, γH2AX levels, fork stalling and cell cycle arrest, reduced the cytotoxic effect of ATR inhibition, improved DNA repair and prevented the protein carbonylation in XP-V cells irradiated with UVA. The mutagenesis by UVA-light was also investigated by exome DNA sequencing of cellular clones. The data indicated a significative increase of mutagenesis in XP-V irradiated cells compared to control cells, and the identification of mutation types indicated a high increase of C>T transitions, probably as result of error-prone replication of pyrimidine dimers. Nevertheless, the induction of C>A transversions were also detected, probably due to oxidized DNA damage. Curiously, when XP-V and control cells were compared, in the absence of irradiation, these transversions were also detected, maybe due to endogenous oxidation of DNA. In addition, in silico analyses showed that UVA-irradiated XP-V cells had a mutation signature similar to the observed for skin cancer. The data demonstrate XP-V cells are sensitive to UVA-light and DNA damage, including by oxidative stress, trigger cell responses and induce mutagenesis in these patients. Therefore, besides showing that UVA-irradiation may generate deleterious effects in the skin of XP-V patients, the data also contribute to understand how these light wavelengths may damage human cells in general
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Étude protéomique des partenaires d`interaction de XPA en présence et en absence de dommage à l`ADNSekheri, Meriem S. 01 1900 (has links)
La réparation par excision de nucléotides (NER) permet l'élimination des lésions provoquant une distorsion de la double hélice de l’ADN. Ces lésions sont induites par plusieurs agents environnementaux comme les rayons UV, ainsi que par certaines drogues chimio- thérapeutiques tel que le cisplatine. Des défauts dans la NER conduisent à de rares maladies autosomiques héréditaires : La xérodermie pigmentaire (XP), le syndrome de Cockayne (CS), le syndrome de sensibilité aux UVSS et la trichothiodystrophie (TTD). Ces maladies sont associées soit à une prédisposition élevée au cancer de la peau et / ou à de graves anomalies du développement neurologique. Le groupe de patients XP-A représente le deuxième groupe (XP) le plus fréquent, et possède la forme la plus sévère combinant cancer de la peau avec un haut risque de dégénérescence neurologique. À date, aucune explication n`a été proposée pour les symptômes neurologiques observés chez ces patients. Nous avions suggéré ainsi que la protéine XPA possède d`autres fonctions dans d`autres processus cellulaires, ceci en interagissant avec des partenaires protéiques différents de ceux déjà connus. Afin de confirmer cette hypothèse nous avions réalisé une étude protéomique à grande échelle en combinant la spectrométrie de masse à une immunoprécipitation en Tandem d`affinité (TAP), afin d`identifier de nouvelles protéines interagissant directement avec XPA. Nous avions montré que XPA peut interagir avec MRE11, la protéine clé de la réparation par recombinaison homologue. Des études additionnelles sont requises pour confirmer cette interaction et comprendre sa fonction / To maintain genome integrity and ensure the continuation of transcription, helix distorting DNA lesions induced by UV and other environmental mutagens are eliminated through a highly-versatile DNA repair pathway: nucleotide excision repair (NER). Mutations in 11 genes (XPC, XPE, XPB, XPD, XPG, XPA, XPG, TTD-A, CSA, CSB and UVSSA), among the 30 genes directly involved in NER, have been associated with the human genetic disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS), trichothiodystrophy (TTD), and UV-sensitive syndrome (UVSS). Patients of these syndromes display a wide variety of clinical features that range from normal development with extreme predisposition to cancer, to neurodevelopmental defects associated with premature aging abnormalities. The connection between DNA damage and neurodegeneration remains unclear, i.e. cannot be explained by a DNA-repair deficiency alone, implying that various repair factors perform other functions beyond the repair process. XP-A is the second most common form of XP. XP-A cells have very low levels of NER activity and are sensitive to killing by UV light. It is one of the most severely affected XP groups, with the onset of cutaneous features, skin cancer, ocular features, and severe early onset neurological disease. Therefore we hypothesize that XPA interacts with cellular proteins that regulate its functions either in UV damage repair or in neurological development. To test this, our major aim was to carry out a large-scale proteomics investigation to identify novel interacting partners for XPA in the absence or presence of genotoxic stress, thus providing clues on the origins of neurodegeneration observed in many XP-A patients. We provide evidence that XPA can interact with MRE11, the key factor in repair of double strand breraks by homologous Recombination. Future experiments will be aimed at determining the impact of the XPA/MRE11 interaction functions in cells.
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