The replication-timing program constitutes a key element of the organization and coordination of numerous nuclear processes in eukaryotes. This program is established at a crucial moment in the cell cycle and occurs simultaneously with the organization of the genome, thus indicating the vital significance of this process. With recent technological achievements of high-throughput approaches, a very strong link has been confirmed between replication timing, transcriptional activity, the epigenetic and mutational landscape, and the 3D organization of the genome. There is also a clear relationship between replication stress, replication timing, and genomic instability, but the extent to which they are mutually linked to each other is unclear. Recent evidence has shown that replication timing is affected in cancer cells, although the cause and consequence of this effect remain unknown. However, in-depth studies remain to be performed to characterize the molecular mechanisms of replication-timing regulation and clearly identify different cis- and trans-acting factors. The results of these studies will potentially facilitate the discovery of new therapeutic pathways, particularly for personalized medicine, or new biomarkers. This review focuses on the complex relationship between replication timing, replication stress, and genomic instability.

DNA replication must be faithful and follow a well-defined spatiotemporal program closely linked to transcriptional activity, epigenomic marks, intranuclear structures, mutation rate and cell fate determination. Among the readouts of the spatiotemporal program of DNA replication, replication timing analyses require not only complex and time-consuming experimental procedures, but also skills in bioinformatics. We developed a dedicated Shiny interactive web application, the START-R (Simple Tool for the Analysis of the Replication Timing based on R) suite, which analyzes DNA replication timing in a given organism with high-throughput data. It reduces the time required for generating and analyzing simultaneously data from several samples. It automatically detects different types of timing regions and identifies significant differences between two experimental conditions in ∼15 min. In conclusion, START-R suite allows quick, efficient and easier analyses of DNA replication timing for all organisms. This novel approach can be used by every biologist. It is now simpler to use this method in order to understand, for example, whether ‘a favorite gene or protein’ has an impact on replication process or, indirectly, on genomic organization (as Hi-C experiments), by comparing the replication timing profiles between wild-type and mutant cell lines.

CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference.

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.