[SAMHD1 acts at stalled replication forks to prevent interferon induction]. / SAMHD1 agit sur les fourches de réplication bloquées pour empêcher l'induction d'interféron.

DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are frequently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. This slowing down, known as replicative stress, plays a central role in tumour development. Complex processes, which are not yet fully understood, are set up to respond to this stress. Certain nucleases, such as MRE11 and DNA2, degrade the neo-replicated DNA at the level of blocked forks, allowing the replication to restart. The interferon pathway is a defense mechanism against pathogens that detects the presence of foreign nucleic acids in the cytoplasm and activates the innate immune response. DNA fragments resulting from genomic DNA metabolism (repair, retrotransposition) can diffuse into the cytoplasm and activate this pathway. A pathological manifestation of this process is the Aicardi-Goutières syndrome, a rare disease characterized by chronic inflammation leading to neurodegenerative and developmental problems. In this encephalopathy, it has been suggested that DNA replication may generate cytosolic DNA fragments, but the mechanisms involved have not been characterized. SAMHD1 is frequently mutated in the Aicardi-Goutières syndrome as well as in some cancers, but its role in the etiology of these diseases was largely unknown. We show that cytosolic DNA accumulates in SAMHD1-deficient cells, particularly in the presence of replicative stress, activating the interferon response. SAMHD1 is important for DNA replication under normal conditions and for the processing of stopped forks, independent of its dNTPase activity. In addition, SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. When SAMHD1 is absent, degradation of neosynthesized DNA is inhibited, which prevents activation of the replication checkpoint and leads to failure to restart the replication forks. Resection of the replication forks is performed by an alternative mechanism which releases DNA fragments into the cytosol, activating the interferon response. The results obtained show, for the first time, a direct link between the response to replication stress and the production of interferons. These results have important implications for our understanding of the Aicardi-Goutières syndrome and cancers related to SAMHD1. For example, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments that trigger the inflammatory response in cells deficient for SAMHD1. We can therefore imagine that blocking the activity of these enzymes could decrease the production of DNA fragments and, ultimately, the activation of innate immunity in these cells. In addition, the interferon pathway plays an essential role in the therapeutic efficacy of irradiation and certain chemotherapeutic agents such as oxaliplatin. Modulating this response could therefore be of much wider interest in anti-tumour therapy.

La réplication de l'ADN est un processus extrêmement complexe, impliquant des milliers de fourches de réplication progressant le long des chromosomes. Ces fourches sont fréquemment ralenties ou arrêtées par différents obstacles, tels que des structures secondaires de l'ADN, des protéines agissant sur la chromatine ou encore un manque de nucléotides. Ce ralentissement, qualifié de stress réplicatif, joue un rôle central dans le développement tumoral. Des processus complexes, qui ne sont pas encore totalement connus, sont mis en place pour répondre à ce stress. Certaines nucléases, comme MRE11 et DNA2, dégradent l'ADN néorépliqué au niveau des fourches bloquées, ce qui permet le redémarrage des réplisomes. La voie interféron est un mécanisme de défense contre les agents pathogènes qui détecte la présence d'acides nucléiques étrangers dans le cytoplasme et active la réponse immunitaire innée. Des fragments d'ADN issus du métabolisme de l'ADN génomique (réparation, rétrotransposition) peuvent diffuser dans le cytoplasme et activer cette voie. Une manifestation pathologique de ce processus est le syndrome d'Aicardi-Goutières, une maladie rare caractérisée par une inflammation chronique générant des problèmes neurodégénératifs et développementaux. Dans le cadre de cette encéphalopathie, il a été suggéré que la réplication de l'ADN pouvait générer des fragments d'ADN cytosoliques, mais les mécanismes impliqués n'avaient pas été caractérisés. SAMHD1 est fréquemment muté dans le syndrome d'Aicardi-Goutières ainsi que dans certains cancers, mais son rôle dans l'étiologie de ces maladies était jusqu'à présent largement inconnu. Nous montrons que de l'ADN cytosolique s'accumule dans les cellules déficientes pour SAMHD1, particulièrement en présence de stress réplicatif, activant la réponse interféron. Par ailleurs, SAMHD1 est important pour la réplication de l'ADN en conditions normales et pour le processing des fourches arrêtées, indépendamment de son activité dNTPase. De plus, SAMHD1 stimule l'activité exonucléase de MRE11 in vitro. Lorsque SAMHD1 est absent, la dégradation de l'ADN néosynthétisé est inhibée, ce qui empêche l'activation du checkpoint de réplication et entraine un défaut de redémarrage des fourches de réplication. De plus, la résection des fourches de réplication est réalisée par un mécanisme alternatif qui libère des fragments d'ADN dans le cytosol, activant la réponse interféron. Les résultats obtenus montrent, pour la première fois, un lien direct entre la réponse au stress réplicatif et la production d'interférons. Ces résultats ont des conséquences importantes dans notre compréhension du syndrome d'Aicardi Goutières et des cancers liés à SAMHD1. Par exemple, nous avons démontré que MRE11 et RECQ1 sont responsables de la production des fragments d'ADN qui déclenchent la réponse inflammatoire dans les cellules déficientes pour SAMHD1. Nous pouvons donc imaginer que bloquer l'activité de ces enzymes pourrait diminuer la production des fragments d'ADN et, in fine, l'activation de l'immunité innée dans ces cellules. Par ailleurs, la voie interférons joue un rôle essentiel dans l'efficacité thérapeutique de l'irradiation et de certains agents chimiothérapiques comme l'oxaliplatine. Moduler cette réponse pourrait donc avoir un intérêt beaucoup plus large en thérapie anti-tumorale.

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain.

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.

Molecular dissection of interactions between Rad51 and members of the recombination-repair group.

Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.

Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1.

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.

Homomeric interaction of the mouse Rad52 protein.

The Rad52 protein plays a crucial role in repairing DNA damage and homologous recombination, possibly by virtue of its ability to catalyze annealing of single-stranded DNA. In agreement with recent genetic data, we here present results based on the two-hybrid system, demonstrating that mouse Rad52p is able to form homomeric complexes. A small domain necessary and sufficient for the self-interaction is located in the conserved N-terminus of the protein. These data contribute to the important insights into the architecture of the multi-protein complex involved in recombinational DNA repair.

Targeted deletions created in yeast vectors by recombinational excision.

We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.

Money as a tool to extinguish conditioned responses to cocaine in addicts.

Classical conditioning principles offer a nondrug way to treat cocaine dependence. Eleven male subjects with the primary diagnosis of cocaine dependence were placed into one of two groups. The experimental group was asked to handle $500 cash in a mock budgetary task. The control group was asked to just imagine handling and budgeting the money. The subjects rated their craving-related feelings before and after each task. The experimental group showed significantly more craving after the money-handling task as compared to the control group, and the scores improved with time and as more tasks were completed. These data show that craving induced by handling cash is powerful and can be attenuated, at least on a short-term basis, using classical extinction procedures.

Dynamics of eye fundus changes in two types of experimental brain oedema.

During the experimentally induced cerebral oedema and intracranial hypertension the eye fundus changes have been evaluated in generally anaesthetized rats. Cerebral oedema was caused either by means of an osmotic or cryogenic method. Dynamics of the observed ocular changes were indirectly correlated with the origin, development and regression of the cerebral oedema caused by both different etiologic factors.