Remodelage de la chromatine par des nano moteurs dépendants d’ATP et réparation de bases de l’ADN – CROREMBER

In eukaryotic cells DNA is packaged into chromatin. Cells use remodeling chromatin factors to overcome the general repression associated with the nucleosomal organization. Energy-dependent nucleosome remodeling factors disrupt histone-DNA interactions at the expense of ATP hydrolysis. Chromatin remodeling factors are complexes consisting of a dedicated ATPase associated with several additional subunits. It was firmly established that the chromatin remodelers exhibit a DNA translocase activity. All the types of remodeling ATPases can induce nucleosome 'sliding' but the reason of the observed directionality within symmetrical core particles remains obscure. Besides, SWI/SNF-related remodeling factors can disrupt histone-DNA interactions more extensively such that eviction of a histone octamer from DNA can be observed. Despite many efforts the mechanisms by which the remodeling factors act remain unclear. Interestingly, chromatin remodelers were unable to mobilize the mH2A and H2A.Bbd histone variant nucleosomes. In contrast, we recently found that nucleolin enhances the remodeling capacity of both SWI/SNF and ACF, ie exhibit a co-remodeling activity. It would be of primary interest to find the origin of this co-remodeling activity and to identify other protein factors exhibiting such activity. In this project we propose to study the structural transitions within the nucleosomes induced by the remodeling complexes SWI/SNF, RCS, ISWI and ACF. In particular, we focus on the mechanisms of action of these chromatin remodeling complexes. We aim to understand why and how the histone variants macroH2A and H2A.Bbd impede the functioning of the remodeling complexes. The mechanism of the histone chaperones (nucleolin etc) - mediated enhancement of remodeler-induced nucleosome mobilization is also addressed together with trying to identify other protein factors exhibiting co-remodeling activity and studying their functioning. Genomic DNA is prone to tremendous number of insults by a myriad of endogenous and exogenous factors. The base excision repair (BER) is the major mechanism used by the cell for the excision of the oxidatively generated DNA damage. Evidences were provided that defects in DNA repair and increased genetic instability are linked to cancer proneness. The proteins involved in BER are well characterized and the different steps in BER are reconstituted in vitro on naked DNA templates by using either purified or recombinant factors. In contrast to naked DNA templates, the mechanism of BER with DNA wrapped on nucleosome remains very poorly studied and elusive. Importantly, reports of BER on more physiological templates such as nucleosomal arrays are missing, the main reason being the lack of chromatinized template suitable for addressing this question. No experiments are published aiming to understand the dynamics of 8-oxoG (the most frequently induced damage repaired by BER) recognition and removal from either nucleosomes or nucleosomal arrays. The reported data on both the involvement and role of histone variants and histone chaperones in BER are very scarce. This project is focused on the role of chromatin structure and dynamics in the repair of 8-oxoG, the most common oxidative base damage. In vitro studies with reconstituted nucleosomes or nucleosomal arrays containing a single (8-oxoG) lesion at distinct positions within a selected individual nucleosome will be carried out and the efficiency of the BER will be measured. The role of the linker histone H1, histone variants, ATP chromatin remodeling machines and histones chaperones exhibiting chromatin co-remodeling activities on the repair of 8-oxoG and clustered lesions will be investigated. We also propose to elucidate how OGG1 and other glycosylases recognize the site of DNA damage in both naked DNA and chromatin templates. To this end we will use our novel UV laser protein-DNA crosslinking and photofootpring technique developed in our labs, which allow 'visualizing' the dynamics of protein-DNA interaction with 'snapshots' of few milliseconds and at one nucleotide resolution. The expected information is crucial for the elucidation the mechanisms of DNA repair in physiological templates. This is an interdisciplinary project, based on original combination of state of the art physical and molecular biology methodologies to study very hot and important biological problems. The elucidation of the mechanism of action of the chromatin remodelers, its impediment by some histone variants and the proteins with co-remodeling activity will be of crucial importance for the understanding of the control of transcription, DNA repair, DNA replication and recombination and many other cell-cycle dependent and vital for the cell processes. Some of the expected information is unique in terms that it cannot be obtained by the currently used approaches.