Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase

XRCC1, un élément clef de la réparation des dommages de l'ADN couplée à la réplication

XRCC1 est un facteur central des voies de réparation des cassures simple brin (SSBR) ou des bases endommagées (BER). Il organise un réseau complexe d interactions protéiques avec les acteurs de la réparation grâce à ses domaines BRCT1 et BRCT2. Le domaine BRCT1 interagit avec l'enzyme qui détecte et signale les cassures dans l'ADN, PARP-1, ainsi qu'avec le poly(ADP-ribose) (PAR), permettant son recrutement rapide au site de dommage. Afin de mieux comprendre les fonctions du domaine BRCT1 de XRCC1 dans la réparation des dommages de l ADN, nous avons cherché à isoler de nouveaux partenaires par une approche protéomique s'appuyant sur la spectrométrie de masse. Nous avons identifié deux nouveaux partenaires : la sous unité catalytique de la protéine kinase DNA-PK impliquée dans la réparation de l'ADN par recombinaison non homologue (NHEJ) et la sous unité p58 du complexe ADN polymérase a-primase impliqué dans la replication de l'ADN. XRCC1 interagit avec DNA-PK et stimule son activité in vitro de phosphorylation de la sérine 15 de p53. En retour, XRCC1 est phosphorylé par DNA-PK au niveau de sa sérine 371 en réponse à une exposition aux rayonnements X et cette phosphorylation régule la transition entre une forme monomère et une forme dimère de XRCC1 et est requise pour la réparation efficace des casssures double brin in vitro. Le mutant non phosphorylable XRCC1-S371L perd sa capacité à stimuler DNA-PK, et induit un défaut de réparation des cassures double brin induites par des rayonnements X. Nos résultats suggèrent que XRCC1 pourrait recruter DNA-PK pour engager la voie de réparation NHEJ lorsqu'une cassure simple-brin a été convertie en cassure double-brin lors de la phase S. XRCC1 interagit par son domaine BRCT1 avec la sous-unité p58 du complexe replicatif Pol a-primase et les deux protéines co-localisent in vivo. Nous montrons que p58 lie le PAR ce qui entraîne l inhibition de l activité primase. La surexpression du domaine BRCT1 de XRCC1 dans des cellules HeLa induit l hétéromodification du domaine BRCT1 surexprimé et l accumulation des cellules en début de phase S après traitement par un agent alkylant. Nous montrons que ce blocage de la réplication à lieu entre la formation des complexes de pré-initiation et le recrutement de PCNA, et qu elle est dépendante de la synthèse de PAR Ce travail révèle une nouvelle fonction de XRCC1 et du PAR, qui est de réguler l'initiation de la réplication lorsque l'ADN est endommagé. L ensemble de nos résultats décrivent XRCC1 et PARP-1 comme des éléments centraux de la coordination entre réparation et réplication de l ADN, ainsi que dans la transition entre SSBR et DSBR. Lors de la réplication d un ADN endommagé, le couple XRCC1/PARP-1, via le PAR porté par le domaine BRCT1 de XRCC1, freine l avancement de la fourche de réplication en interagissant avec p58, inhibant de la sorte l ADN primase. Ceci permettrait à la cellule de réparer les lésions présentes sur l ADN afin d éviter la collision entre la fourche de réplication et une cassure simple-brin de l ADN non réparée. Néanmoins, en cas de conversion du SSB en DSB, XRCC1 stimule l activité de DNA-PK afin de promouvoir la réparation du DSB.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation.

International audienceSIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-)(/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer