Interplay between DNA N-glycosylases/AP lyases at multiply damaged sites and biological consequences

Evidence has emerged that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. The first repair event occurring at a multiply damaged site (MDS) is of major importance and will largely contribute to the hazardousness of MDS. Here, using protein extracts from wild type or hOGG1-overexpressing Chinese hamster ovary cells, we investigated the initial incision rate at base damage and the formation of repair intermediates in various complex MDS. These MDS comprise a 1 nt gap and 3–4 base damage, including 8-oxoguanine (oG) and 5-hydroxyuracil (hU). We report a hierarchy in base excision that mainly depends on the nature and the distribution of the damage. We also show that excision at both oG and hU, and consequently DSB formation, can be modulated by hOGG1 overexpression. Anyhow, for all the MDS analyzed, DSB formation is limited, due to impaired base excision. Interestingly, repair intermediates contain a short single-stranded region carrying a potentially mutagenic base damage. This in vitro study provides new insight into the processing of MDS and suggests that repair intermediates resulting from the processing of such MDS are rather mutagenic than toxic

Effets du rayonnement ultraviolet a sur la réplication de l'adn chez les eucaryotes supérieurs

Le rayonnement ultraviolet (UV) émis par le soleil et qui atteint la peau de chaque individu est composé majoritairement de photons UVA ( de 315 à 400 nm), le reste (5 à 10 %) étant composé d UVB les plus longs ( de 300 à 315 nm), car les radiations de longueur d onde 300nm, c est-à-dire les plus toxiques en terme de santé humaine, sont absorbées par la couche d ozone stratosphérique. Contrairement aux UVB, les radiations UVA sont faiblement absorbées par l ADN et de fait, génèrent peu de dimères cyclobutaniques de pyrimidines. Néanmoins, un des problèmes majeurs posés par une exposition aux UVA tient à ce que ce rayonnement excite certains composés endogènes photosensibles, inducteurs de la production d espèces réactives de l oxygène (ROS) qui peuvent alors endommager les composants cellulaires tels que les lipides,les acides nucléiques et les protéines. De ce fait, si les UVB restent le facteur étiologique majeur contribuant à la cancérogenèse cutanée photoinduite, un rôle des UVA, via la production de ROS, semble également émerger. Des précédents travaux obtenus au laboratoire ont montré que le rayonnement UVA ralentit la réplication de l ADN, indépendamment de l activation des points de contrôle du cycle cellulaire. Les auteurs ont émis l hypothèse que les UVA, via l oxydation des protéines, pouvaient altérer la machinerie de réplication. Mon travail de thèse a donc consisté à tenter de préciser le mécanisme qui gouverne ce retard de la réplication de l ADN induit par les UVA dans les cellules de mammifères.Pour étudier au niveau moléculaire les effets des UVA sur la réplication, nous avons tout d abord mis en place et utilisé au laboratoire la technique du peignage moléculaire (DNA combing) qui permet de mesurer divers paramètres de la réplication. Ainsi, nous montrons que le rayonnement UVA inhibe immédiatement et transitoirement les vitesses de fourches alors que l inhibition sur l initiation des origines est plus prolongée. Dans le cadre d une collaboration, nous montrons également que les radiations UVA induisent une diminution modeste et transitoire du pool de dNTPs intracellulaires. La complémentation en ribonucléosides ne semble pas suffisante pour restaurer une vélocité normale de fourches immédiatement après UVA, ni la réplication dans sa totalité. En parallèle, nous observons l oxydation réversible de la sous-unité R1 de la ribonucléotide réductase impliquée dans la biosynthèse des dNTPs. Bien que cette oxydation ne puisse expliquer la baisse transitoire du pool de nucléotides après UVA, nous ne pouvons pas exclure que d autres formes d oxydation de la RNR puissent affecter son activité.La présence d azide de sodium (NaN3) au cours de l irradiation UVA prévient le retard réplicatif, limite l oxydation de la sous-unité R1 et la diminution du pool de dNTPs, ce qui démontre que ce retard de réplication est totalement dépendant des ROS, principalement de l oxygène singulet généré pendant l irradiation.L ensemble de nos résultats indiquent que les UVA affectent le processus de réplication en modifiant non seulement la vélocité des fourches mais également l initiation des origines de réplication. Puisqu une perturbation de la réplication est une cause majeure d instabilité génétique, il reste à déterminer si, dans nos conditions expérimentales, les radiations UVAfavorisent cette instabilité. Enfin, nous pensons que la ou les cibles des ROS induites par les UVA sont essentiellement cytosoliques et que le mécanisme conduisant à l inhibition de la réplication n est pas spécifique de ces ROS mais pourrait s observer en utilisant d autres types de stress oxydant.The solar UV radiation that reaches the earth s surface is composed of 10 % UVB (280 320 nm) and 90 % UVA (320 400 nm) the main toxic radiations (wavelengths below 300 nm) being blocked by the stratospheric ozone. Unlike UVB, the UVA component of solar radiation is weakly absorbed by DNA. Nevertheless, one of major problems due to UVA exposure is the production of reactive oxygen species (ROS) through the interaction with endogenous and exogenous chromophores. These ROS cause damage to DNA, lipids and proteins. Even if UVB remains the major etiological factor known to be implicated in photoinduced cutaneous carcinogenesis, a novel role for UVA via the production of ROS seems to emerge. In our lab, previous works have provided evidence that exposure of mammalian cells to UVA-induced ROS led to delayed S-phase and reduced DNA synthesis, by a yet unknown process, which does not require a functional DNA damage checkpoint response, despite ATM-, ATR-, p38-dependent pathways activation. The authors proposed that inhibition of DNA replication is due to impaired replication fork progression and/or origins activation, as a consequence of UVA-induced oxidative damage to proteins rather than to DNA. The project for my PhD thesis is to better understand the mechanism underlying this UVA-induced slowdown of DNA replication in human cells.To study at the molecular level the effects of UVA on DNA replication, we used the DNA combing methodology. This technique allows measurement of the fork velocity and of the origins density. We show that UVA-induced ROS inhibit immediately after irradiation, but transiently, the progression of replication forks, while the inhibition on the initiation of originslasts longer. By HPLC-MS, we show that UVA radiation induces a moderate and transient decrease of the level of each intracellular dNTP. The supply of ribonucleosides doesn t seem to be sufficient to restore neither a normal forks velocity immediately post-UVA nor the overall slowdown of DNA replication. In addition, we observe a reversible oxidation of the subunit R1 of ribonucleotide reductase, an enzyme which is involved in dNTPs biosynthesis. This oxidation cannot explain the transient reduction of dNTPs pool after UVA exposure, but other types of RNR oxidative modification could affect its activity. During UVA irradiation, the presence of the antioxidant sodium azide (NaN3) prevents the delay of DNA replication, limits the oxidation of the subunit R1 and the decrease of dNTPs pool. These results strongly suggest that the slowdown of DNA replication totally depends on ROS, in particular on singlet oxygen production induced by UVA.Altogether, our data indicate that UVA irradiation affects the process of DNA replication by modifying the forks velocity and the activation of origins. As DNA replication impairment is a major cause of genetic instability, it is of importance to determine if UVA irradiation leads to this instability in our experimental conditions. Finally, we suspect that the target of UVAinduced ROS is essentially cytosolic and that the mechanism driving the inhibition of replication is not specific of UVA-induced ROS, but could be also observed with other types of oxidative stress.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

CONSEQUENCES BIOLOGIQUES D'UNE EXPOSITION SOLAIRE. QUANTIFICATION ET REPARATION DES LESIONS BIPYRIMIDIQUES INDUITES PAR LES RADIATIONS ULTRAVIOLETTES DANS L'ADN GENOMIQUE DE CELLULES DE MAMMIFERE. ROLE DES UVA DANS LA MUTAGENESE SOLAIRE

PARIS-BIUSJ-Physique recherche (751052113) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Radiation-induced clustered DNA lesions: Repair and mutagenesis.

Clustered DNA lesions, also called Multiply Damaged Sites, is the hallmark of ionizing radiation. It is defined as the combination of two or more lesions, comprising strand breaks, oxidatively generated base damage, abasic sites within one or two DNA helix turns, created by the passage of a single radiation track. DSB clustered lesions associate DSB and several base damage and abasic sites in close vicinity, and are assimilated to complex DSB. Non-DSB clustered lesions comprise single strand break, base damage and abasic sites. At radiation with low Linear Energy Transfer (LET), such as X-rays or γ-rays clustered DNA lesions are 3-4 times more abundant than DSB. Their proportion and their complexity increase with increasing LET; they may represent a large part of the damage to DNA. Studies in vitro using engineered clustered DNA lesions of increasing complexity have greatly enhanced our understanding on how non-DSB clustered lesions are processed. Base excision repair is compromised, the observed hierarchy in the processing of the lesions within a cluster leads to the formation of SSB or DSB as repair intermediates and increases the lifetime of the lesions. As a consequence, the chances of mutation drastically increase. Complex DSB, either formed directly by irradiation or by the processing of non-DSB clustered lesions, are repaired by slow kinetics or left unrepaired and cause cell death or pass mitosis. In surviving cells, large deletions, translocations, and chromosomal aberrations are observed. This review details the most recent data on the processing of non-DSB clustered lesions and complex DSB and tends to demonstrate the high significance of these specific DNA damage in terms of genomic instability induction

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

International audienc

Unravelling UVA-induced mutagenesis.

International audienceUltraviolet A (UVA) radiation represents more than 90% of the solar UV radiation reaching Earth's surface. Exposure to solar UV radiation is a major risk in the occurrence of non-melanoma skin cancer. Whole genome sequencing data of melanoma tumors recently obtained makes it possible also to definitively associate malignant melanoma with sunlight exposure. Even though UVB has long been established as the major cause of skin cancer, the relative contribution of UVA is still unclear. In this review, we first report on the formation of DNA damage induced by UVA radiation, and on recent advances on the associated mechanism. We then discuss the controversial data on the UVA-induced mutational events obtained for various types of eukaryotic cells, including human skin cells. This may help unravel the role of UVA in the various steps of photocarcinogenesis. The connection to photocarcinogenesis is more extensively discussed by other authors in this issue