Les facteurs endogènes et exogènes induisent diverses modifications chimiques et structurales dans l’ADN cellulaire et sont à l’origine de nombreuses erreurs lors de la réplication et de la division cellulaire. Elles sont souvent à l’origine de cancers et de maladies chroniques liées à l’âge. Les agents alkylants induisent des lésions complexes de l’ADN. Ils sont utilisés comme des outils efficaces dans les traitements par chimiothérapies. Les deux caractéristiques majeures des lésions complexes de l’ADN sont d’une part l’encombrement et d’autre part la présence de plus d’une modification dans un tour d’hélice. Les données biochimiques et génétiques montrent que l’élimination des dommages complexes de l’ADN nécessitent plusieurs voies de réparation distinctes. Les caractéristiques cliniques des maladies héréditaires caractérisées par des désordres de réparation de l’ADN, comme l’anémie de Fanconi et le syndrome de Cockayne, visent la nature complexe des lésions oxydatives endogène incluant les adduits encombrants et les pontages interbrins (PIBs). D’autre part, les effets biologiques sévères des agents ionisants, des traitements de chimiothérapie et des cancérigènes environnementaux sont impliqués dans la formation de cassures double brins, des PIBs et des adduits encombrants. Bien que ces derniers représentent une faible proportion des dommages de l’ADN induit par stress oxydatif et les agents chimiques dans les cellules, ils sont extrêmement cytotoxiques s’ils ne sont pas réparés. Par exemple, les cellules cancéreuses sont très sensibles aux PIBs et aux cassures double brins. Alors que la consommation de produits contenant de l’acide aristolochique (AA), qui génère des adduits encombrants dans l’ADN cellulaire, peut être à l’origine de neuropathies et a été associé à une cancérogénèse plus élevée que la fumée de cigarette. L’objectif majeur de ce projet est l’étude de la réparation des lésions complexes de l’ADN et leur implication dans le développement des cancers et leur thérapie. Dans cette présente étude, nous nous sommes intéressés aux mécanismes moléculaires de la réparation des PIBs et des adduits encombrants de l’ADN et leurs possibles rôles dans la chimio- et radiorésistance acquise. De plus, nous avons étudié le mécanisme moléculaire impliqué dans la mutagénèse induite par la réparation aberrante des adduits encombrants initiée par des ADN glycosylases de la voie BER dans des cellules bactériennes et de mammifères. Durant les trois années de thèse, (i) j’ai construit et caractérisé différents substrats d’ADN contenant diverses bases oxydées, des PIBs induits par le psoralène, des adduits encombrants aristolactame et des photoproduits issus des UV, (ii) j’ai purifié divers ADN glycosylases recombinantes (TDG, MBD4, NEIL1 et NEIL3), (iii) j’ai reconstruit in vitro la réparation des lésions de l’ADN et (iv) j’ai également étudié les interactions protéine-protéine et les modifications post-traductionnelles des protéines impliquées dans la réparation des PIBs générés par le cisplatine initiée par les ADN glycosylases NEIL1 et NEIL3. En combinant les différentes approches développées dans notre laboratoire, nous avons identifié et caractérisé des voies de réparation alternatives impliquées dans l’élimination des dommages complexes de l’ADN. Les résultats obtenus dans ce travail permettraient de comprendre la mécanistique du développement de certains cancers et à identifier les facteurs associés à la chimio- radiorésistance des cellules cancéreuses et par conséquent contribuer au développement de nouvelles préventions de stratégies thérapeutiques. / Endogenous and exogenous mutagenic factors induce variety of chemical and structural modifications in cellular DNA that are at the origin of errors that occur during DNA replication and cell division and often give rise to cancer and other age-related chronic diseases. Importantly, alkylating agents which induce complex DNA lesions are used as a powerful tool for anti-cancer chemotherapy. Two most important features of complex DNA lesions are their bulky character and presence of more than one modification within one turn of DNA helix. Genetic and biochemical data indicate that the elimination of complex DNA lesions requires several distinct DNA repair pathways. The clinical features of inherited human DNA repair deficient disorders such as Fanconi anemia and Cockayne syndrome point to complex nature of endogenous oxidative DNA damage which include bulky adducts and inter-strand DNA crosslinks (ICLs). On the other hand, severe biological effects of ionizing radiation, anticancer drugs and environmental carcinogens are correlated with formation of dirty DNA strand breaks, ICLs and bulky adducts. Although complex lesions typically constitute relatively small fraction of the total DNA damage induced by oxidative stress and drugs in cells, they are extremely cytotoxic if not repaired. For example, cancer cells are primarily very sensitive to ICLs and dirty DNA strand breaks. While, consumption of products containing aristolochic acid (AA), which generates bulky adenine DNA adducts in cellular DNA, can cause neuropathy and has been associated with higher risk of cancer than cigarette smoking. The major objective of the present project is to study the repair of complex DNA lesions and their implications in cancer development and therapy. Here, we investigated molecular mechanisms of the repair of ICLs and bulky DNA lesions and their possible role in the acquired chemo-resistance of cancer cells. In addition, we studied the molecular mechanism of mutagenesis induced by the aberrant DNA glycosylase-mediated BER towards bulky adducts in bacterial and mammalian cells. During three years of the project, I have (i) constructed and characterized DNA substrates containing various oxidized bases, psoralen-derived ICLs, bulky aristolactam-adenine adducts and UV photoproducts; (ii) purified several recombinant human DNA glycosylases (TDG, MBD4. NEIL1 and NEIL3); (iii) reconstituted in vitro the repair of complex DNA lesions; and finally (iv) studied the protein-protein interactions and post-translational protein modifications involved in the DNA glycosylases NEIL1 and NEIL3 initiated repair of cis-platinum induced ICLs. By combining the approaches developed in our laboratories we have identified and characterized alternative DNA repair pathways involved in the cellular processing of complex DNA damage. The results obtained in present work can provide mechanistic understanding of the development of certain cancer and lead to identification of the factors associated with the acquired chemo- and radio-resistance of tumour cells and therefore would contribute to development of new prevention and therapeutic strategies.

No description available.

No description available.

Les oxydations sur les bases nucléiques constituent l’une des sources principale d’apparition de lésions sur l’ADN, qui peuvent être mutagènes ou létales pour les cellules en l’absence de réparation de l’ADN. La Formamidopyrimidine-ADN glycosylase (Fpg), une enzyme procaryote du système de réparation de l’ADN par excision de base (BER), initie la réparation d’un large panel de lésions de ce type via ses activités ADN glycosylase (excision de la base oxydée) et AP lyase (clivage du site abasique par ß,d-élimination). Nous avons réalisé des études fonctionnelles par des techniques biochimiques et structurales par cristallographie des rayons X afin de préciser la spécificité de substrat et le mécanisme catalytique de Fpg. Ainsi, nous avons pu mettre en évidence des déterminants structuraux permettant à cette enzyme d’accommoder des lésions de tailles très différentes dans son site actif, en l’occurrence des résidus 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) substitués ou non en N7 par des adduits encombrants. D’autre part, nous avons caractérisé structuralement et fonctionnellement la reconnaissance et l’excision par Fpg d’une lésion pyrimidique, la 5-hydroxy-5-méthyle-hydantoïne (Hyd). Ainsi, nous avons montré que cette lésion appariée à une cytosine était un bon substrat pour l’enzyme, et nous avons précisé structuralement le mode de reconnaissance de l’Hyd par Fpg. D’autre part, nous avons mis en évidence un comportement inattendu de l’enzyme sur ce substrat. En l’occurrence, nous avons montré biochimiquement et structuralement qu’un pontage covalent se formait en quantités non négligeables entre Fpg et l’Hyd dans des conditions physiologiques. / Oxidations on nucleic bases constitute one of the major sources of DNA lesions appearance, which can be mutagenic or lethal for cells in the absence of DNA repair. The prokaryotic Formamidopyrimidine-DNA glycosylase (Fpg), a base excision DNA repair (BER) enzyme, initiate the repair of a wide range of such lesions via its DNA glycosylase (excision of the oxidized base) and AP lyase (cleavage of the AP site by ß,d-elimination) activities. We carried out functional studies by biochemical techniques and structural studies by X-ray crystallography so as to state Fpg’s substrate specificity and catalytic mechanism. Thus, we have been able to underline the structural determinants enabling this enzyme to accommodate lesions of very different sizes in its active site, in this case 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) residues N7-substituted or not by bulky adducts. On the other hand, we structurally and functionally characterized the recognition and excision by Fpg of a pyrimidic lesion, the 5-hydroxy-5-methyl-hydantoin (Hyd). Thus, we have shown that this lesion paired with a cytosine was a good substrate for the enzyme, and stated structurally the recognition mode of Hyd by Fpg. On the other hand, we have underlined an unexpected behaviour of the enzyme on this substrate. In this case, we have biochemically and structurally shown that a covalent link was formed in sizeable quantities between Fpg and Hyd in physiological conditions.

Objectives: The aim of this study was to determine if exposure to volatile organic compounds can lead to DNA damage and impaired DNA repair capacity. Nail cosmetics is a fast growing industry around the world where employees and clients are subjected to various chemical substances which may be harmful to their health: such as formaldehyde, toluene, acetone, xylene, ethylmethacrylate, methylmethacrylate and n–buthyl acetate. These chemicals have the potential to be harmful to their health and exposure to these chemicals should be actively controlled. Formaldehyde is classified as a human carcinogen by the IARC, whereas, toluene and xylene are group three carcinogens, classified in 1999 (not classified as carcinogenic to humans), and various studies have linked DNA damage and impaired DNA repair to the above mentioned substances. Methods: Fifteen nail technicians were monitored by means of personal air sampling, measuring formaldehyde, toluene, xylene, acetone and ethylmethacrylate exposure. Fifteen unexposed subjects were chosen and matched for age and smoking habits with the exposed group. Heparinised blood samples were obtained from each test subject with which the Comet Assay was performed on lymphocytes to determine DNA damage and repair ability. Results: Exposure to ethylmethacrylates and methylmethacrylates leads to DNA damage. Methylmethacrylate causes DNA damage by specifically targeting pyrimidine (fpg) bases. N–buthyl acetate, xylene and acetone exposure impaired DNA repair capacity. The exposed group showed signs of Class III and Class IV DNA damage, whereas the control group had little Class III damage and no indication of Class IV damage. The overall DNA repair ability of the nail technicians was slightly impaired when compared to that of the control group, which is in concurrence with previous studies. Smoking habits and age did not show significant influences on the level of DNA damage and repair when compared with the control group. Conclusion: Exposure to volatile organic compounds such as ethylmethacryale and methylmethacrylate may lead to DNA damage and altered DNA repair in some individuals, although further studies are recommended. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2011.

Objectives: The aim of this study was to determine if exposure to volatile organic compounds can lead to DNA damage and impaired DNA repair capacity. Nail cosmetics is a fast growing industry around the world where employees and clients are subjected to various chemical substances which may be harmful to their health: such as formaldehyde, toluene, acetone, xylene, ethylmethacrylate, methylmethacrylate and n–buthyl acetate. These chemicals have the potential to be harmful to their health and exposure to these chemicals should be actively controlled. Formaldehyde is classified as a human carcinogen by the IARC, whereas, toluene and xylene are group three carcinogens, classified in 1999 (not classified as carcinogenic to humans), and various studies have linked DNA damage and impaired DNA repair to the above mentioned substances. Methods: Fifteen nail technicians were monitored by means of personal air sampling, measuring formaldehyde, toluene, xylene, acetone and ethylmethacrylate exposure. Fifteen unexposed subjects were chosen and matched for age and smoking habits with the exposed group. Heparinised blood samples were obtained from each test subject with which the Comet Assay was performed on lymphocytes to determine DNA damage and repair ability. Results: Exposure to ethylmethacrylates and methylmethacrylates leads to DNA damage. Methylmethacrylate causes DNA damage by specifically targeting pyrimidine (fpg) bases. N–buthyl acetate, xylene and acetone exposure impaired DNA repair capacity. The exposed group showed signs of Class III and Class IV DNA damage, whereas the control group had little Class III damage and no indication of Class IV damage. The overall DNA repair ability of the nail technicians was slightly impaired when compared to that of the control group, which is in concurrence with previous studies. Smoking habits and age did not show significant influences on the level of DNA damage and repair when compared with the control group. Conclusion: Exposure to volatile organic compounds such as ethylmethacryale and methylmethacrylate may lead to DNA damage and altered DNA repair in some individuals, although further studies are recommended. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2011.

Les dommages oxydatifs de l’ADN sont impliqués dans les processus pathologiques que sont le cancer, les maladies neurodégénératives ou le vieillissement. Ces dommages résultent en partie de l’action des espèces réactives de l’oxygène (ERO), qui proviennent du métabolisme cellulaire ou d’agents exogènes (physiques ou chimiques), et qui conduisent à différents types de lésions parmi lesquelles l’oxydation des bases de l’ADN (8-oxoguanine, 8-oxoG) ou la formation de sites abasiques AP (apurique/apyrimidique). Ces lésions, qui si elles ne sont pas éliminées conduisent à des processus de mutagenèse ou de mort cellulaire, sont prises en charge spécifiquement par le système de réparation de l’ADN par excision de base ou BER. Le BER est initié par l’action d’une ADN glycosylase, telles que la 8-oxoG-ADN glycosylase (Ogg1) chargée d’éliminer la 8-oxoG, une lésion très abondante. Une étude par « double-hybride » initiatrice de ce projet a révélé l’existence d’une interaction in vivo chez S. cerevisiae entre la protéine yOgg1 et la sous-unité catalytique de l’ADN polymérase réplicative Polε (yPol2), également impliquée dans la voie BER chez la levure. Nos travaux démontrent que yOgg1 et yPol2 interagissent bien physiquement entre elles et de façon spécifique. Une étude par troncations et mutagenèse dirigée nous a permis d’identifier le domaine 3’→5’ exonucléase de yPol2 comme faisant partie de la forme tronquée minimale de yPol2 capable d’interagir avec yOgg1. La poche du site actif de yOgg1 et/ou son voisinage immédiat pourrait contenir pour partie le site d’interaction pour yPol2. Nous observons d’ailleurs une corrélation nette entre l’activité de yOgg1 et sa capacité à interagir avec yPol2 dans la levure. De même, l’activité 3’→5’ exonucléase de yPol2 pourrait être liée à son interaction avec yOgg1. D’un point de vue fonctionnel, yPol2 stimulerait l’activité AP lyase de yOgg1 et le couplage entre l’activité ADN glycosylase et AP lyase de l’enzyme, permettant ainsi une meilleure coordination de l’étape d’excision du nucléoside endommagé et l’étape de resynthèse de l’ADN dans la voie BER. / Oxidative DNA damages are involved in pathological processes such as cancer, neurodegenerative diseases and aging. Part of these damages results from the action of reactive oxygen species (ROS), which are produced by cellular metabolism or (physical or chemical) exogenous agents. They lead to different types of DNA lesions including DNA base oxidation (8-oxoguanine, 8-oxoG) and abasic site formation (AP, apuric/apyrimidic). If not removed, these lesions lead to mutagenesis or cell death. Most of base lesions are dealt specifically by the base excision repair (BER) pathway. BER is initiated by a DNA glycosylase, such as 8-oxoG-DNA glycosylase (Ogg1) which is responsible for the removal of 8-oxoG. In previous unpublished work, a yeast two-hybrid study revealed the existence in S. cerevisiae of an interaction between yOgg1 and the catalytic subunit of the replicative DNA polymerase Polε (yPol2), also involved in the BER pathway in eukaryotes. Our work shows that yOgg1 and yPol2 physically and specifically interact with each other. Truncation and site-directed mutagenesis studies allowed us to identify the 3 ' → 5' exonuclease activity domain of yPol2 as part of the minimal form of yPol2 still able to interact with yOgg1. The active site of yOgg1 and/or its immediate vicinity may contain part of its interaction domain with yPol2. Besides, we observe a clear correlation between yOgg1 catalytic activity and its ability to interact with yPol2 in vivo. Similarly, the 3'→5' exonuclease activity of yPol2 could be useful to its interaction with yOgg1. From a functional point of view, yPol2 stimulates in vitro the AP lyase activity of yOgg1 and the coupling of both DNA glycosylase and AP lyase enzyme activity. The interaction yOgg1/yPol2 could allow a better coordination of damaged nucleoside excision and DNA re-synthesis steps in BER.

The thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined. The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed. Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point.