DynaRep

The efficient and accurate duplication of eukaryotic genomes depends on the sequential firing of thousands of replication origins distributed along the chromosomes. Origin activation follows a temporal program that is imposed by the chromosomal context and is controlled by S-phase checkpoints. The correct execution of this replication program is essential for the maintenance of genome integrity during DNA replication. Indeed, replication fork progression is frequently challenged by DNA lesions and by a variety of natural pause sites. Arrested forks are unstable structures, which represent a major threat for the integrity of the genome if they are not promptly stabilized and restarted. Replication stress activates the Mec1/ATR checkpoint pathway, which prevents the collapse of stalled forks and represses the activation of late replication origins. Activation of this surveillance pathway has been extensively studied in situations where the cells are exposed to genotoxic drugs. However, the mechanisms by which it coordinates origin activation, fork progression and fork recovery remains poorly understood. Moreover, the mechanism by which Mec1/ATR promotes the correct execution of the replication program in normal growth conditions remains unclear. The aim of this project is to provide a genome-wide view of the execution of the replication program in normal growth conditions and in cells exposed to genotoxic stress. We propose to follow two major strategies. We will determine how the S-phase checkpoint modulates origin usage and fork progression in S. cerevisiae cells exposed to hydroxyurea. These studies should clarify two major unresolved issues regarding the mechanisms by which cells detect replication stress and modulate fork progression. We will also determine how these mechanisms operate in a normal S phase by performing a genome-wide analysis of the sequences that induce spontaneous replication fork pausing in yeast and in human cells. We will use the combination of powerful techniques both at a genome-wide (ChIP-on-chip, ChIP-Seq) and single-molecule (DNA combing) levels to appreciate the complexity and variability of DNA replication programs from a cell to another. In the first part of the project, we will clarify two important aspects of the DNA replication response that remain poorly understood. The first issue concerns the mechanism by which cells detect arrested forks. We recently identified new mediators of the replication checkpoint pathway and propose to further characterize their function in this critical process. We will also characterize an unexpected implication of the DNA damage checkpoint in the regulation of replication timing. The second issue concerns the regulation of replication fork speed by the replication checkpoint, which is currently a subject of controversy. To clarify this point, we will analyze fork rates in a variety of replication mutants using single-molecule and genome-wide approaches, in the presence or the absence of replication stress. A key point will be to monitor the dNTPs pools which appear to be highly regulated during S phase and during the checkpoint response. The second part of this project aims at determining whether S-phase checkpoints operate in a normal S phase and whether they contribute to the correct execution of the replication program. Indeed, replication fork progression is frequently challenged by DNA lesions and by a variety of natural pause sites, including condensed chromatin structures, non-histone nucleoprotein complexes, and highly-expressed genes. We plan to identify regions of the genome that are intrinsically difficult to replicate, using a ChIP-on-chip analysis of a DNA damage marker known to be activated by checkpoint pathways at stalled forks. Interplay between transcription and replication will also be assessed, since it appears as a potential source of genome instability, for instance at highly transcribed genomic regions. Replication and transcription are globally altered in cells overexpressing oncogenes, we will therefore also concentrate on the study of interferences between these two processes at a genome-wide level. Recent evidence indicates that spontaneous replication stress occurs spontaneously in precancerous lesions and promotes the development of cancer. Identifying the origin of this replication stress would represent a major step towards a better understanding of the early stages of tumorigenesis.