Evidence for a Rad18-Independent Frameshift Mutagenesis Pathway in Human Cell-Free Extracts

Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF)-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G) and 5'-GGCGCC-3' (NarI site), induces –1 and –2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion

Mutagenese induite par une amine aromatique cancerogene et sa reparation chez E. coli

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Modulation of transcription reveals a new mechanism of triplet repeat instability in Escherichia coli

International audienc

Expansion of CTG repeats from human disease genes is dependent upon replication mechanisms in Escherichia coli: the effect of long patch mismatch repair revisited

International audienceMany human hereditary disease genes have been recently associated with the expansion of CTG/GAC repeats. We have used a plasmid-based assay in Escherichia coli to investigate the instability of a (CTG/GAC) insert containing 64 repeats. Using this assay, expansions were biochemically detected and subsequently quantified. We show that the occurence of expansions within these trinucleotide repeats is dependent upon replicative mechanisms. Expansions of up to 30 repeats and deletions of almost all possible sizes occured regardless of the orientation of the insert relative to the replication origin. In contradiction to a previous report, the mismatch repair pathway was found to strongly stabilize these repeat stretches

Simulation numérique de répliques de zéolithes en carbone (structures et propriétés d'adsorption en vue d'une application au stockage d'hydrogène)

Le stockage d hydrogène est le verrou technologique pour l utilisation de ce vecteur énergétique dans domaine du transport automobile. Les matrices poreuses en carbone sont des matériaux qui pourraient permettre de réaliser un stockage efficace. Nous avons étudié des nanostructures à porosité contrôlée, répliques carbonées de silices mésoporeuses ou de zéolithes. Nous simulons par une approche statistique les répliques de quatre zéolithes à l échelle atomique : AlPO4-5, silicalite, FAU et EMT. Nous montrons que les faujasites induisent des structures de nanotubes interconnectés, formant des réseaux de pores nanométriques interconnectés, avec de très bonnes propriétés mécaniques pour de très faibles densités de matériaux. Nous montrons que toute phase purement carbonée ne sera d aucun intérêt à température ambiante en comparaison avec des réservoirs classiques. Par dopage au lithium, ces matériaux peuvent en théorie stocker à 300 bars autant d hydrogène qu un système classique fonctionnant à 700 bars. Les résultats des capacités théoriques permettent d envisager une application embarquée dans le domaine du transport automobile.Hydrogen storage is the key issue to envisage this gas as an energy vector in the field of transportation. Porous carbons are materials that are considered as possible candidates. We have studied well-controlled microporous carbon nanostructures, carbonaceous replicas of mesoporous ordered silica materials and zeolites. We realized numerically the atomic nanostructures of the carbon replication of four zeolites : AlPO4-5, silicalite, and Faujasite (FAU and EMT). The faujasite replicas allow nanocasting a new form of carbon cristalline solid made of tetrahedrally or hexagonally interconnected nanotubes. The pore size networks are nanometric giving to these materials optimized hydrogen molecular storage capacities. However, we demonstrate that these new carbon forms are not interesting for a room temperature efficient storage compared to the void space of a classical tank. We showed that doping with alkaline element such as lithium one could store the same quantities at 300 bars than a classical tank at 700 bars. This result is a possible route to achieve interesting performances for on-board docking systems.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli

International audienc

RecA-Dependent DNA Repair Results in Increased Heteroplasmy of the Arabidopsis Mitochondrial Genome

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution