The duplication and segregation of chromosomes are two fundamental events in cell reproduction. Any mistakes in these processes can lead to genome instability and contribute to tumour progression. Eukaryotic chromosomes are replicated from numerous origins, which are formed in G1 and activated throughout S phase according to specific spatio-temporal programs. We use the yeast S. cerevisiae and mouse embryonary fibroblasts (MEFs) as model systems to understand how these programs are set-up, carried out and what they are good for. They appear as primary determinants of chromosome structure, expression, cohesion and stability.
Keywords : cancer, cell cycle, DNA replication, genome instability, yeast genetics.
We are interested in how eukaryotes succeed in duplicating their large genomes accurately and completely during the imparted time before mitosis. This is possible because DNA synthesis begins from a large number of sites (330 replication origins in yeast, >10,000 in metazoans) distributed fairly uniformly along chromosomes and activated throughout S phase according to a predefined pattern. Future origins are formed, i.e., assemble pre-replicative complexes, during the G1 phase of the cell cycle, by a process that is antagonized by cyclin-dependent kinase (CDK) activity. We showed that the yeast CDK inhibitor Sic1 is key to prevent precocious S-CDK activation and to allow origin licensing in late G1. Without Sic1, cells begin S phase from fewer origins, do not complete replication in time and undergo chromosome breaks and rearrangements in early mitosis.
Current work is aimed at understanding why sic1∆ mutants, or any other situation that modifies origin usage, generate such high rates of genomic instability. It is generally believed that cells do not enter mitosis unless they have completed DNA replication, but this deserves reinvestigation. We are testing this notion directly by changing the temporal control and length of S phase, as well as by using genetic screens. We are also studying genetically defined murine primary fibroblasts to see if changes in replication patterns precede and might be causative of their genomic instability.
Even in budding yeast where autonomous replication sequences (ARS) are well defined, most origins do not fire every cell cycle, a feature usually occulted by population-based techniques. We have developed methods to label nascent DNA in live yeast with BrdU, followed by its detection and mapping on single DNA molecules displayed on microscope glass slides by a process called DNA combing. Thanks to new software developed in the lab, the analysis of DNA combing images is now more rigorous and amenable to studying replication dynamics at high-throughput in mammalian cells.