hnRNP K: An HDM2 Target and Transcriptional Coactivator of p53 in Response to DNA Damage

SummaryIn response to DNA damage, mammalian cells trigger the p53-dependent transcriptional induction of factors that regulate DNA repair, cell-cycle progression, or cell survival. Through differential proteomics, we identify heterogeneous nuclear ribonucleoprotein K (hnRNP K) as being rapidly induced by DNA damage in a manner that requires the DNA-damage signaling kinases ATM or ATR. Induction of hnRNP K ensues through the inhibition of its ubiquitin-dependent proteasomal degradation mediated by the ubiquitin E3 ligase HDM2/MDM2. Strikingly, hnRNP K depletion abrogates transcriptional induction of p53 target genes and causes defects in DNA-damage-induced cell-cycle-checkpoint arrests. Furthermore, in response to DNA damage, p53 and hnRNP K are recruited to the promoters of p53-responsive genes in a mutually dependent manner. These findings establish hnRNP K as a new HDM2 target and show that, by serving as a cofactor for p53, hnRNP K plays key roles in coordinating transcriptional responses to DNA damage

A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation.

BACKGROUND: The mammalian DNA-damage response (DDR) has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs), mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. METHODS: In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+) with the MDM2 antagonist, Nutlin-3. RESULTS: Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37). Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. CONCLUSION: To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist

Etude du mécanisme de la recombinaison homologue chez les rétrovirus

Un des facteurs majeurs générant la variabilité génétique chez les rétrovirus est la recombinaison homologue. La recombinaison homologue est le résultat d'un transfert de la synthèse de l'ADN, au cours de la transcription inverse, d'un ARN génomique (ARN donneur) à l'autre ARN (ARN accepteur) présent dans le virion. Afin de disséquer le mécanisme de transfert de brin, nous avons développé un système expérimental qui permet d'étudier la recombinaison sur des séquences rétrovirales in vitro. Un tiers du génome du VIH-1 a été analysé et nous avons montré que des transferts de brin se produisaient fréquemment sur la plupart des séquences étudiées. Nous avons mis en évidence, pour la première fois, un rôle des régions avoisinantes sur la capacité recombinogène d'une région donnée. La capacité de recombinaison d'une séquence donnée est dépendante de la présence d'une structure en tige-boucle. Cette structure est plutôt requise sur l'ARN accepteur que sur le donneur. De plus, nous avons montré que la réaction de transfert est une réaction qui passe par un intermédiaire mono-moléculaire dans lequel un complexe constitué par la transcriptase inverse (RT), l'ARN donneur, l'ADN naissant et l'ARN accepteur se forme avant la réalisation du transfert. Sur la base de ces résultats, nous avons construit un modèle original du transfert de brin. Dans ce modèle, une hybridation de l'accepteur par sa structure tige-boucle sur l'ADN naissant permet de rapprocher l'accepteur du complexe RT/ARN donneur/ADN et d'exercer une contrainte mécanique sur l'ADN qui induit une perturbation de la transcription inverse sur le donneur et aboutit à un remplacement de celui-ci par l'accepteur. Il est bien connu que le repliement de l'ARN est impliqué dans plusieurs processus biologiques, nous avons montré qu'elle est aussi impliquée dans le phénomène qui participe à la génération de la variabilité génétique chez les rétrovirus: la recombinaison homologue.Homologous recombination is one of the main factors generating genetic variability in retrovirus. Recombination results from a transfer, by the reverse transcriptase, of the DNA synthesis from a genomic RNA (donor RNA) to the other one (acceptor RNA) presents within the virion. In order to dissect the molecular mechanism of this phenomenon, we have developed a reconstituted in vitro system, which allowed us to study recombination on any retroviral sequence of interest. One third of the genome has been investigated and most regions analysed yielded a high degree of recombination. We have identified, for the first time, that recombination efficiency of a given sequence was dependent on its folding and namely on the presence of a stable hairpin. This hairpin structure was required to be present in the acceptor RNA. Furthermore, we have shown that the strand transfer reaction is an intramolecular process where the acceptor RNA is complexed to the nascent DNA before the transfer. Based on these results, we have proposed a model of recombination in which the acceptor RNA, by its hairpin structure, hybridises to the nascent DNA before the switch occurs. This hybridisation both allows the acceptor RNA to be in close proximity with the nascent DNA and disrupts the process of reverse transcription ongoing on the donor RNA, thereby leading to a displacement of the donor RNA from the polemerase active site and its replacement by the acceptor RNA. It is well known that the folding of the RNA is involved in several biological processes, we have now demonstrated that it is also involved in the process, which generates genetic variability in retrovirus: homologous recombination.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer

Design, synthesis, chemical characterization, biological evaluation, and docking study of new 1,3,4-oxadiazole homonucleoside analogs

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