L'ADN étant le support de l'information génétique, les lésions de l'ADN provoquées par différents stress physiques ou chimiques sont un défi pour les systèmes de réparation cellulaire. Parmi ceux-ci le système de réparation par excision de bases (BER) implique plusieurs enzymes dont les objectifs sont la reconnaissance et le retrait de la base lésée, fonctions bien connues pour deux glycosylases : Fpg Procaryote et OGG1 Eucaryote. De nombreuses approches ont été décrites pour étudier les interactions ADN/protéine in vitro. Avec la résonance plasmonique de surface par imagerie (SPRi), nous disposons d'une technique d'analyse en temps réel, sans marquage avec laquelle nous avons pu observer des interactions parallélisées d'une même protéine enzymatique purifiée (Fpg, OGG1, EndoIV ou Ape1) vis-à-vis de différentes lésions sur des oligonucléotides de synthèse immobilisés sur une surface d'or. Les dommages étudiés sont une base oxydée (8-oxoG), une base cyclisée (cycloadénine) et des analogues de sites abasiques (THF et C3). Nous avons également étudié l'action de ces mêmes enzymes sur des lésions multiples, en tandem, associant les bases 8-oxoG et 8-oxoA sur le même brin d'ADN. L'originalité de notre dispositif associe l'analyse directe de l'interaction ADN/protéine et l'approche indirecte de sa conséquence par une stratégie d'hybridation et d'amplification du signal après une rampe thermique. Les résultats obtenus permettent d'envisager l'utilisation de notre technique pour observer la réparation simultanée de certaines lésions par des extraits cellulaires pour des travaux de biochimie ou des extraits tissulaires humains pour des travaux de biologie médicale.

No description available.

<p>Ageing is associated with a progressive decline in cognitive and physical function, as well as neurodegeneration. The DNA damage theory of ageing postulates that phenotypes associated with chronological ageing result from a time dependent accumulation of DNA damage caused by endogenously generated reactive oxygen species (ROS). In this work, we have used a host cell reactivation (HCR) technique to examine base excision repair (BER), the major pathway for removal of ROS generated damage, in fibroblasts from normal individuals and from patients with Cockayne syndrome (CS). The HCR assay utilizes an adenovirus encoded β-galactosidase (β-gal) reporter gene treated with methylene blue plus visible light (MB+VL) to measure BER of 7,8-dihydro-8-oxoguanine (8-oxoG). The results presented here demonstrate that host cell repair mechanisms remove MB+VL generated 8-oxoG from viral DNA and that reactivation of gene expression correlates with cellular repair capacity and requires CSA and CSB. Using the HCR assay, we demonstrate that culturing of primary human fibroblasts in media containing low levels of MB increases BER, suggesting increased DNA repair capacity may play a role in the therapeutic application of MB in Alzheimer’s disease treatment. We also demonstrate that BER decreases <em>in vitro </em>with increasing number of cell divisions, and that HCR of the damaged reporter gene is lower in fibroblasts from older donors. Using a second β-gal reporter gene assay, the enhanced expression assay, we were unable to show a relationship between the degree of decreased BER in CS and severity of clinical phenotype. However, we identified an interaction between CSB and the telomere protein TRF2. Overexpression of TRF2 leads to decreased nucleotide excision repair of UVC induced damage in a CSB dependent manner. We also demonstrate defective telomeres in the absence of functional CSB. The data presented in this work provide additional support for the DNA damage theory of ageing.</p> / Doctor of Philosophy (PhD)

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2′-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and 13C- and 15N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.

No description available.

The low redox potential of guanines (G 1.29 V vs. NHE) compared to other nucleobases, makes them potentially susceptible to attack by exogenous and endogenous damaging species. This property of guanine has also been utilized for the development of several anticancer agents including the well-known platinum complexes, cisplatin and carboplatin. The two closely related nickel complexes, NiCR and NiCR-2H, exhibit significant differences in cytotoxicity towards MCF-7 cancer cells. In the first part of this work, we explain this difference using biochemical and biophysical approaches to study their interactions with duplex DNA. The nickel complexes were found to selectively oxidize guanines in bulged DNA structures in the presence of oxidant and notably NiCR-2H oxidizes guanines more efficiently than NiCR. According to 1 H NMR studies, NiCR-2H binds strongly to the N7 position of dGMP compared to NiCR and could be an important oxidation product of NiCR under physiological conditions. The second part of this work focuses on the secondary DNA structures known as G-quadruplex formed in the guanine rich telomeric region. G-quadruplex is formed by stacking of G-quartets (a coplanar cyclic array of four Gs) on top of each other. Its formation is known to inhibit the activity of the reverse transcriptase telomerase that is overexpressed in 80-90% cancer cells. The guanines in telomeric DNA are readily oxidized due to their low redox potential and the major oxidation product is 7, 8-dihydro-8-oxoguanine (OxodG). OxodG (0.58 V vs. NHE) can further be oxidized in the presence of one electron oxidants and the resulting product forms adducts with endogenous nucleophiles such as spermine. In light of these findings, we hereby designed and synthesized novel bifunctional perylene derivatives that can selectively bind to the telomeric DNA via G-quadruplex formation and subsequently react with OxodG in close proximity. These compounds have strong binding affinity towards G-quadruplex and can significantly stabilize the OxodG containing G-quadruplex motif by end stacking on the upper G-quartet. The effect of these compounds on telomerase activity and cytotoxicity towards Hep3B cancer cells was also evaluated.

No description available.

The expanding therapeutic use of thalidomide (TD) remains limited by its species-specific teratogenicity in humans and rabbits, but not rodents. The R and S isomers of TD may be selectively responsible for its respective therapeutic and teratogenic effects, but rapid in vivo racemization makes this impossible to confirm. Fluorothalidomide (FTD), a fluorinated TD analogue with stable, non-racemizing isomers, may serve as a model compound for determining stereoselective effects. In vivo, FTD was undetectable in plasma, suggesting rapid breakdown, as confirmed in vitro, where FTD hydrolyzed up to 22-fold faster than TD. Unlike TD, FTD in pregnant rabbits and mice was highly toxic and lethal to both dams and fetuses. In rabbit embryo culture, FTD initiated optic (eye) vesicle and hindbrain but not classic limb bud embryopathies. Chemical instability, potent general toxicity and absence of limb bud embryopathies make FTD an unsuitable stereoselective model for TD teratogenesis. TD teratogenesis may involve its bioactivation by embryonic prostaglandin H synthases (PHSs) to a free radical intermediate that increases embryopathic reactive oxygen species (ROS) formation. However, the teratogenic potential of rapidly formed TD hydrolysis products and the determinants of species-specific teratogenesis are unclear. For some teratogens, mouse strains that are resistant in vivo are susceptible in embryo culture, suggesting maternal and/or placental determinants of risk. However, TD and two hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaraic acid (PGA), were non-embryopathic in CD-1 mouse embryo culture. Also, mice deficient in oxoguanine glycosylase 1 (OGG1), which repairs oxidatively damaged DNA, were resistant to TD embryopathies in culture and in vivo. Therefore, murine resistance to TD teratogenesis is dependent on embryonic factors, rather than maternal/placental determinants or increased DNA repair. In contrast, rabbit embryos exposed in culture to TD, PGMA and PGA exhibited head/brain, otic (ear) vesicle and classic limb bud embryopathies, validating the first mammalian embryo culture model for TD teratogenesis and providing the first evidence of a teratogenic role for TD hydrolysis products. Pretreatment with eicosatetraynoic acid (ETYA), a dual PHS/lipoxygenase inhibitor, or phenylbutylnitrone (PBN), a free radical spin trapping agent, completely blocked TD, PGMA and PGA-initiated embryopathies, implicating a PHS-dependent, ROS-mediated embryopathic mechanism.