Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Déficience dans la réparation par excision de nucléotides en phase S induite par la séquestration du facteur de réplication RPA

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants.

Urinary 8-oxo-2′-deoxyguanosine:Redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2′-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G → T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically. © 2002 Elsevier Science Inc.

Deoxycytidine glyoxal:Lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p = .001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p = .0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected. © 2003 Elsevier Science Inc.

Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 59-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 39-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase b and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Étude du comportement de la chromatine, de la régulation de la transcription et réparation des gènes de l’ARNr avant la réplication de l’ADN et assemblage de la réparation par excision de nucléotides chez Saccharomyces cerevisiae

Résumé : Le nucléole est considéré comme étant une « usine » à produire des ribosomes. Cette production est la fonction la plus énergivore de la cellule. Elle met en jeu les trois ARN polymérases et représente 80% de l’activité de transcription au sein d’une cellule. Les trois quarts de cette activité de transcription correspondent à la synthèse des ARNr par l’ARN polymérase I (ARNPI). Ainsi mieux comprendre les mécanismes cellulaires se déroulant à l’intérieur de ce compartiment permettra le développement de nouveaux traitements contre le cancer. La synthèse d’ARNr par l’ARNPI est régulée à trois niveaux : l’initiation de la transcription, l’élongation et le nombre de gènes de l’ARNr en transcription. La plupart des travaux qui se sont intéressés à ces niveaux de régulation ont été réalisés avec des cellules en phase exponentielle de croissance. Au cours de mes travaux, je me suis attardé sur la régulation de la transcription par l’ARNPI au cours de la phase G1 du cycle cellulaire et au début de la phase S. Ainsi mes résultats ont montré que si la chromatine des gènes de l’ARNr est essentiellement dépourvue de nucléosomes, la régulation de l’ARNPI diffère dans des cellules en G1 et au début de la phase S. J’ai pu de ce fait observer qu’en G1, la transcription de l’ARNPI se concentre sur un nombre réduit de gènes en transcription. Dans des cellules arrêtées au début de la phase S avec de l’hydroxyurée, la transcription de l’ARNPI est perturbée par un défaut de maturation de l’ARNR. Fort de ces résultats sur la nature des gènes ribosomaux en phase G1, je me suis attardé à la réparation de ces gènes lors de cette phase. Alors que dans des cellules en phase exponentielle de croissance irradiées avec des UVC, la chromatine des gènes de l’ARNr se ferme ; je n’ai pas observé la formation de nucléosomes suite à l’irradiation de cellules synchronisée en G1. Mes résultats montrent également que la réparation est plus efficace. Parallèlement, j’ai exploré l’assemblage du complexe de réparation par excision de nucléotides. Toutefois, les résultats obtenus sont peu concluants.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2. CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m-2) of UVB radiation.

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

XPC polymorphisms play a role in tissue-specific carcinogenesis: a meta-analysis

XPC participates in the initial recognition of DNA damage during the DNA nucleotide excision repair process in global genomic repair. Polymorphisms in XPC gene have been analyzed in case-control studies to assess the cancer risk attributed to these variants, but results are conflicting. To clarify the impact of XPC polymorphisms in cancer risk, we performed a meta-analysis that included 33 published case-control studies. Polymorphisms analyzed were Lys939Gln and Ala499Val. The overall summary odds ratio (OR) for the associations of the 939Gln/Gln genotype with risk of cancer was 1.01 (95% confidence interval (95% CI): 0.94-1.09), but there were statistically significant associations for lung cancer, observed for the recessive genetic model (Lys/Lys + Lys/Gln vs Gln/Gln), (OR 1.30; 95% CI: 1.113-1.53), whereas for breast cancer a reduced but nonsignificant risk was observed for the same model (OR 0.87; 95% CI: 0.74-1.01). The results for Ala499Val showed a significant overall increase in cancer risk (OR 1.15; 95% CI: 1.02-1.31), and for bladder cancer in both the simple genetic model (Ala/Ala vs Val/Val) (OR 1.30; 95% CI: 1.04-1.61) and the recessive genetic model (Ala/Ala + Ala/Val vs Val/Val) (OR 1.32; 95% CI: 1.06-1.63). Our meta-analysis supports that polymorphisms in XPC may represent low-penetrance susceptibility gene variants for breast, bladder, head and neck, and lung cancer. XPC is a good candidate for large-scale epidemiological case-control studies that may lead to improvement in the management of highly prevalent cancers.

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Susceptibilidade genética para cancro da mama

RESUMO: O cancro da mama é a patologia oncológica mais frequente nas mulheres sendo o responsável pela maior taxa de mortalidade por cancro no sexo feminino. Contudo, as causas inerentes a esta patologia permanecem por esclarecer. Nos últimos anos tem-se verificado que o risco para patologia neoplásica depende de factores ambientais e genéticos, estando estes últimos associados à variabilidade genética inter-individual. Polimorfismos genéticos em genes envolvidos no metabolismo de hormonas sexuais, de cancerígenos ambientais e na reparação da lesão genética, são potenciais candidatos a estarem associados à susceptibilidade individual para esta patologia. Assim, neste trabalho desenvolveram-se estudos de associação caso-controlo na população Portuguesa, com vista a avaliar-se o papel atribuído aos polimorfismos na susceptibilidade para cancro da mama. Foram seleccionados polimorfismos em genes envolvidos em diferentes vias mecanicistas: destoxificação de cancerígenos, metabolismo de estrogénios, reparação por excisão de bases, reparação por excisão de nucleótidos, reparação mismatch e reparação por recombinação homóloga. Os resultados obtidos revelaram associação entre os seguintes polimorfismos e a susceptibilidade individual para cancro da mama: os dois SNPs estudados no gene XRCC1 (Arg194Trp e Arg399Gln) e o SNP no gene XRCC3 (Thr241Met) após estratificação pelo status menopausico. Mediante estratificação por status de amamentação os SNPs identificados nos genes MnSOD (Val16Ala) e XRCC2 (Arg118His); um SNP no gene MLH3 (Leu844Pro), e por fim como resultado de interacção gene-gene as interacções descritas por MSH3 Ala1045Thr/MSH6 Gly39Glu e MSH4 Ala97Thr/MLH3 Leu844Pro. Os resultados obtidos e apresentados na presente dissertação, revelam que o estudo de polimorfismos pode representar um papel determinante na etiologia do cancro da mama. No entanto, mais estudos envolvendo estes mesmos polimorfismos em populações casuisticamente superiores serão uma mais-valia nos estudos de associação para esta neoplasia. Adicionalmente, a utilização da metodologia de Pools de DNA, poderá ser uma ferramenta útil na pré-selecção dos polimorfismos mais relevantes a estudar, na medida em que permite estimar a frequência alélica de cada SNP numa determinada população.-----------------------------------ABSTRACT: Breast cancer is the most common form of cancer among women, being the responsible for the highest mortality rate from cancer among the female sex. However, the main causes related to this pathology remain unclear. The risk of neoplasic disease has been connected with genetic and environmental factors. In fact, genes and the environment share the stage for most, if not all, common non-familial cancers, and are related to individual susceptibility. Genetic polymorphisms identified in genes encoding enzymes involved in estrogen metabolism, xenobiotics and DNA repair pathways are believed to be candidates for associations with breast cancer. Therefore, it was our intention to develop case-control studies among the Portuguese population, in order to evaluate the potential role of several genetic polymorphisms in breast cancer susceptibility. We selected polymorphisms in genes involved in different pathways: carcinogenic detoxification, estrogen metabolism, base excision repair, nucleotide excision repair, mismatch repair and double strand break repair by homologous recombination. The results obtained revealed potential associations between some polymorphisms studied and individual susceptibility to breast cancer. Regarding this fact, our results suggest the potential involvement of two XRCC1 gene polymorphisms (Arg194Trp and Arg399Gln) and XRCC3 gene polymorphism (Thr241Met) after stratification to menopausal status and after stratification to breastfeeding status an association of MnSOD gene polymorphism (Val16Ala) and XRCC2 (Arg188His) with the disease. The SNP identified in MLH3 gene (Leu844Pro), and the interaction gene-gene described by MSH3 Ala1045Thr/MSH6 Gly39Glu and MSH4 Ala97Thr/MLH3 Leu844Pro were also related to breast cancer susceptibility. The results shown in the present dissertation have revealed the potential role of polymorphisms in breast cancer etiology. However, further studies will be needed with larger populations to confirm these results. Additionally, the use of DNA pools methodology, as a pre-selection tool, could allow the identification of the most relevant polymorphisms to be studied, estimating the allelic frequency of each SNPs in different populations.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and ofXpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

Caractérisation structurale et fonctionnelle des interactions impliquant TFIIH et la machinerie de réparation de l’ADN

La réparation de l’ADN par excision des nucléotides (NER) est un mécanisme capable de retirer une large variété de lésions causant une distorsion de la double hélice, comme celles causées par les rayons ultraviolets (UV). Comme toutes les voies de réparation de l’ADN, la NER contribue à la prévention de la carcinogénèse en prévenant la mutation de l’ADN. Lors de ce processus, il y a d’abord reconnaissance de la lésion par la protéine XPC/Rad4 (humain/levure) qui recrute ensuite TFIIH. Ce complexe déroule l’ADN par son activité hélicase et recrute l’endonucléase XPG/Rad2 ainsi que d’autres protéines nécessaires à l’excision de l’ADN. Lors de son arrivée au site de lésion, XPG/Rad2 déplace XPC/Rad4. TFIIH agit également lors de la transcription de l’ADN, entre autres par son activité hélicase. Outre cette similarité de la présence de TFIIH lors de la transcription et la réparation, il est possible de se demander en quoi les deux voies sont similaires. Nous nous sommes donc intéressés aux interactions impliquant TFIIH et la machinerie de réparation de l’ADN. Nous avons donc entrepris une caractérisation structurale et fonctionnelle de ces interactions. Nous avons découvert que Rad2 et Rad4 possèdent un motif d’interaction en nous basant sur d’autres interactions de la sous-unité Tfb1 de TFIIH. Par calorimétrie à titrage isotherme, nous avons observé que les segments de ces deux protéines contenant ce motif interagissent avec une grande affinité au domaine PH de Tfb1. Le site de liaison de ces segments sur Tfb1PH est très semblable au site de liaison du domaine de transactivation de p53 et au domaine carboxy-terminal de TFIIEα avec Tfb1PH, tel que démontré par résonance magnétique nucléaire (RMN). De plus, tous ces segments peuvent faire compétition les uns aux autres pour la liaison à Tfb1PH. Nous avons aussi démontré in vivo chez la levure qu’une délétion de Tfb1PH crée une sensibilité aux radiations UV. De plus, la délétion de multiples segments de Rad2 et Rad4, dont les segments d’interaction à Tfb1PH, est nécessaire pour voir une sensibilité aux rayons UV. Ainsi, de multiples interactions sont impliquées dans la liaison de Rad2 et Rad4 à TFIIH. Finalement, les structures des complexes Rad2-Tfb1PH et Rad4-Tfb1PH ont été résolues par RMN. Ces structures sont identiques entre elles et impliquent des résidus hydrophobes interagissant avec des cavités peu profondes de Tfb1PH. Ces structures sont très semblables à la structure de TFIIEα-p62PH. Ces découvertes fournissent ainsi un lien important entre la transcription et la réparation de l’ADN. De plus, elles permettent d’émettre un modèle du mécanisme de déplacement de XPC/Rad4 par XPG/Rad2 au site de dommage à l’ADN. Ces connaissances aident à mieux comprendre les mécanismes de maintient de la stabilité génomique et peuvent ainsi mener à développer de nouvelles thérapies contre le cancer.