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Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the distribution of origin of recognition complex (ORC) binding sites by ChIP-seq of two PfORC subunits and mapped active ORIs using NFS and SNS-seq. We show that ORIs lack sequence specificity but are not randomly distributed, and group in clusters. Licensing is biased towards regions of higher GC content and associated with G-quadruplex forming sequences (G4FS). While strong transcription likely enhances firing, active origins are depleted from transcription start sites. Instead, most accumulate in transcriptionally active gene bodies. Single molecule analysis of nanopore reads containing multiple initiation events, which could have only come from individual nuclei, showed a relationship between the replication fork pace and the distance to the nearest origin. While some similarities were drawn with the canonic eukaryote model, the distribution of ORIs in P. falciparum is likely shaped by unique genomic features such as extreme AT-richness-a product of evolutionary pressure imposed by the parasitic lifestyle., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synchronized activation of DNA replication origins induces genetic instability in lymphoma.

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

DNA replication starts with the opening of DNA at sites called DNA replication origins. From the single sequence-specific DNA replication origin of the small Escherichia coli genome, up to thousands of origins that are necessary to replicate the large human genome, strict sequence specificity has been lost. Nevertheless, genome-wide analyses performed in the recent years, using different mapping methods, demonstrated that there are precise locations along the metazoan genome from which replication initiates. These sites contain relaxed sequence consensus and epigenetic features. There is flexibility in the choice of origins to be used during a given cell cycle, probably imposed by evolution and developmental constraints. Here, we will briefly describe their main features., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although some features underlying replication-origin activation in metazoan cells have been determined, little is known about their regulation during metazoan development. Using the nascent-strand purification method, here we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pregastrula), potential cruciform structures and open chromatin are determining factors that establish replication origins. The observed enrichment of replication origins in transcription factor-binding sites and their presence in promoters of highly transcribed genes, particularly operons, suggest that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when embryonic differentiation programs are set, new origins are selected at enhancers, close to CpG-island-like sequences, and at noncoding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.

To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile., (© 2015 Cayrou et al.; Published by Cold Spring Harbor Laboratory Press.)

DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.

In the genome of eukaryotic cells, DNA synthesis is initiated at multiple sites called origins of DNA replication. Origins must fire only once per cell cycle and how this is achieved is now well understood. However, little is known about the mechanisms that determine when and where replication initiates in a given cell. A large body of evidence indicates that origins are not equal in terms of efficiency and timing of activation. Origin usage also changes concomitantly with the different cell differentiation programs. As DNA replication occurs in the context of chromatin, initiation could be influenced by multiple parameters, such as nucleosome positioning, histone modifications, and three-dimensional (3D) organization of the nucleus. This view is supported by recent genome-wide studies showing that DNA replication profiles are shaped by genetic and epigenetic processes that act both at the local and global levels to regulate origin function in eukaryotic cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps., (Copyright © 2012. Published by Elsevier Inc.)

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates.

At each cell division in humans, 30,000-50,000 DNA replication origins are activated, and it remains unclear how they are selected and recognized by replication factors. DNA replication in multicellular organisms must accommodate variations in growth conditions and DNA damage. It must also adapt to changes in chromatin organization associated with cell differentiation and development. The selection of replication origins in metazoans seems to involve multiple choices, with the appropriate answers depending on the identity of the cell or the conditions of growth. This suggests that during evolution, the use of replication origins became more controlled by epigenetic mechanisms affecting chromosome dynamics and expression than by DNA synthesis per se.

During each cell cycle, thousands of DNA replication origins are activated in each cell of a metazoan organism. Although they appear site-specific, their usage and organization are rather plastic. Moreover, no strict sequence specificity has been observed in contrast to bacterial or Saccharomyces cerevisiae DNA replication origins. Epigenetic regulation linked to chromatin structure, chromosome organization, and transcription has been suggested to explain how DNA replication origins are selected and recognized by replication initiation factors. In this paper, we review these epigenetic features and discuss how, during the previous mitosis, chromosomal architecture might prepare DNA replication origins for a new cell cycle.

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

Although proteins involved in DNA replication in yeast have counterparts in multicellular organisms, the definition of an origin of DNA replication and its control in higher eukaryotes might obey to different rules. Origins of DNA replication that are site-specific have been found, supporting the notion that specific DNA regions are used to initiate DNA synthesis along metazoan chromosomes. However, the notion that specific sequences will define origins is still being debated. The variety and complexity of transcriptional programs that have to be regulated in multicellular organisms may impose a plasticity that would not be compatible with a fixed origin simply defined at the sequence level. Such a plasticity would be essential to developmental programs where the control of DNA replication could be more integrated to the control of gene expression than in unicellular eukaryotes.

In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.