[Show abstract][Hide abstract] ABSTRACT: The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about [Formula: see text] predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.
[Show abstract][Hide abstract] ABSTRACT: Replication origins are where pre-replication complexes are assembled during G1 phase. However, only a subset of the origins is actually "fired" to initiate DNA synthesis during S phase. Whereas factors involved in these steps are relatively well understood now, the mechanisms behind the origin specification, the choice of origins to be fired and determination of their timing are still under active investigation. Recent data show that the origin positions as well as the selection of those to be fired may be determined by multiple factors including sequences, chromatin context, epigenetic information, and some specific genomic features, but that the choice is surprisingly plastic and opportunistic. Timing regulation of firing, on the other hand, appears to be related to cell type-specific intrinsic chromatin architecture in nuclei. The conserved Rif1 protein appears to be a major global regulator of the genome-wide replication timing. Replication timing is regulated also by other factors including checkpoint signals, local chromatin structures, timing and quantity of pre-RC formation, and availability of limiting initiation factors.
Seminars in Cell and Developmental Biology 04/2014; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A "mini-ARS screen" identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion.
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