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LRIG Research Projects

Publié le 2 juillet 2018
​​The base excision repair pathway in mammalian cells
The base excision repair (BER) pathway is the main pathway for the repair of DNA base damage and single strand breaks. These kinds of lesions are generated continuously at high levels in every cells due to the normal metabolism, but their number is highly increased in cells exposed to oxidative stress conditions such as those encountered in pathologies including cancer, neurodegenerative diseases and inflammation. High levels of oxidative DNA damage are also produced in cells exposed to genetoxics such as ionizing radiation and a large number of industrial compounds and therapeutic drugs
Thus BER is a major barrier to prevent accumulation of DNA lesions therefore protecting against cell death or mutagenesis. Base excision repair of modified bases is initiated by lesion-specific DNA glycosylases. After oxidative stress, one of the main base lesions formed in DNA is 8-oxoguanine (8-oxoG) that, if left unrepaired, induces mutations potentially involved in cancer and aging. OGG1 is the major DNA glycoylase responsible for the removal of 8-oxoG.

The OGG1 activity generates an abasic site (AP) that is processed by the endonuclease APE1 that gives rise to a single strand break (SSB); DNA polymerase and ligase activities are then required to restore the DNA integrity.
After analysing for many years the way that DNA glycosylases, APE1 and XRCC1 initiate the repair of 8-oxoG or abasic sites, our laboratory is now interested in studying the effect on BER proteins of cellular environmental changes such as oxidative stress or the switch from stem cells to differentiation. 

We have observed that after induction of 8-oxoG in the cellular DNA, OGG1, APE1 and XRCC1 are recruited from a soluble nucleoplasmic fraction to the chromatin, following a kinetics that is in good correlation with the repair rate. Biochemical fractionation as well as confocal microscopy studies revealed that KBrO3-induced BER foci are completely excluded from heterochromatin rich regions, and are preferentially located in less-condensed DNA areas where active transcription takes place. We are presently trying to characterize the molecular mechanisms involved in the recruitment of the BER complexes to euchromatin regions. Results from a high throughput siRNA screening suggest that proteins involved in chromatin remodelling, transcription regulation, and histone modifications have a role in the relocalisation of BER proteins to euchromatin regions.
Localisation of BER complexes into euchromatin regions after induction of oxidative DNA damage. HeLa cells expressing the fusion proteins OGG1-DsRED (red) and XRCC1-GFP (green) were treated with KBrO3, that mainly induces 8-oxoG. Three hours after the treatment, BER proteins become insoluble and associate to those regions in which DNA (stained with DAPI, blue) is less condensed, corresponding to euchromatin regions.​

​Mitochondria being the main producers of reactive oxygen species in the cell,  mitochondrial genome is particularly exposed to oxidation. Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases, tumorigenesis and aging. It is now clear that BER is the main pathway involved in the removal of these lesions. Interestingly, all the proteins involved in BER are encoded by the nuclear genome and the mechanism responsible for the targeting of the translated protein to the nucleus or to the mitochondria have not been clearly established yet. Different mechanism including alternative splicing, alternative transcription start or dual initiation of translation, have been proposed for different BER proteins. We are presently trying to understand what mechanisms regulate the targeting of the DNA glycosylases, in particular OGG1, to the mitochondria and how BER is coordinated in this organelle. 

OGG1 is localized in both the nucleus and the mitochondria. OGG1-FLAG has been transfected into U2OS cells and visualized using an anti-FLAG antibody (visualized with Alexa488, green). Mitochondria are stainied with MitoTracker Deep Red (magenta), and the nuclear DNA is visualized by DAPI staining (blue). 

More recently we have also established that unexpected factors such as the cellular prion protein can stimulate BER and we are now trying to identify the underlying mechanisms and the possible physiological implications of this response. 

​Genetic variability in Helicobacter pylori.
Helicobacter pylori chronically colonises approximately half of the world’s population. Trans​mission between humans is thought to occur via person-to-person contact during childhood. Persistent infection is usually asymptomatic but can evolve in peptic ulcer disease and gastric cancer. Eradication treatments are based on antibiotics utilization, but during the last decade, H. pylori antibiotic resistance has increased dramatically. Up to 30% of patients are not cured after completing their first course of treatment. This augmentation, as well as the success in infection can be explained by the high adaptability of these bacteria. H. pylori is indeed one of the most genetically diverse bacteria and comparison of the sequenced genomes reveals an amazing variability between strains. This genomic variability is the result of high mutation rates, high recombination efficiency, and natural competence, which allows constant and rapid spread of new mutation among the bacterial population. There is therefore a critical balance between the maintenance of the genetic information and the capacity to adapt. 

Since 1999 our laboratory has contributed to the understanding of the DNA metabolism processes defining the genomic plasticity of H. pylori. Initially, the group has analysed both, biochemically and genetically, the Base Excision Repair pathway, unveiling a new family of DNA glycosylases. Since 2005, the LRIG has turned its attention to the study of Homologous Recombination (HR) in H. pylori allowing the identification of missing components of the mediator complexes responsible for the formation of an active RecA nucleofilament. Homologous recombination being the last step required for the integration of exogenous DNA into the bacterial chromosome, the LRIG has also extended its interests to the analysis of natural transformation in this pathogen. H. pylori presents several important differences with the other competent bacteria starting by the proteins involved in its transport across the two membranes to the fact that in this species natural competence is constitutive. By combining genetics, biochemistry and cell biology approaches we aim at understanding the molecular mechanisms by which the incoming DNA is processed first during its passage through the periplasm and then in the cytoplasm to recombine with the host genome.