Replication Origins: Structure and Interactions with Transcription – ROSIT

The aim of this project is to understand how vertebrate cells cope with the fact that replication and transcription share the same substrate, in particular by determining how transcription interferes with the replication initiation program and vice versa. Upon replicative stress, transcription-replication interferences (TRIs) are exacerbated and tend to impair timely replication completion at two major types of fragile sites; (i) sites nested in early replicating domains (early replicating fragile sites: ERFSs), enriched in robust replication origins and in small, highly transcribed genes. These features favor the formation of R-loops, which can impede replisome progression, (ii) sites located in late replicating domains (common fragile sites, CFSs) found in the core of very long and modestly expressed genes characterized by their paucity in initiation events. A generally accepted explanation is that ongoing transcription removes the pre-replication complexes, a mandatory intermediate in origin building. Central to our project is the identification of cis and trans determinants involved in origin specification and of the relationships linking these determinants to the promoters of fragile genes. By studying a model origin in DT40 chicken cells, we have shown that putative G-quadruplex forming sequences (PQSs) contribute to cis specification of origins and, more recently, that a dimeric PQS (two PQSs on the same strand) constitutes an efficient minimal origin. Such dimers are associated with one third of strong initiation sites in the vertebrate genome, showing their importance in establishing the replication program. The aim of our project is (i) to use genome-wide data to identify specific properties of ERFSs, (ii) to use engineered DT40 lines to test how the minimal origin and transcription interfere, globally and in ERFSs and CFSs and (ii) to explore the proteome around this origin, traversed or not by transcription, in neutral context and in ERFSs and CFSs.