Similar Datasets
Project description:Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.
| S-EPMC11410655 | biostudies-literature
Project description:Faithful DNA replication is essential for genome integrity. Under-replicated DNA leads to chromosome segregation defects, which are reportedly common during embryogenesis. However, DNA replication regulation remains poorly understood in early mammalian embryos. Here, we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and discovered an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing (RT) program, and the entire genome replicated gradually and uniformly using extremely slow-moving forks. In 4-cell embryos, a somatic-cell-like RT program commenced abruptly. However, the fork speed was still slow, S-phase was extended, and SLX4 DNA repair foci increased during G2/M, which was followed by a transient increase in chromosome segregation errors. Importantly, live imaging captured longer S-phase of error cells, and the breakpoints identified by single-cell genome sequencing were enriched in late-replicating regions. By the 8-cell stage, forks gained speed, S-phase was no longer extended, and chromosome aberrations disappeared. Thus, a transient period of genomic instability exists during normal mouse development, which is preceded by a fragile S-phase lacking the coordination between replisome-level regulation and megabase-scale RT regulation, implicating the importance of their coordination for genome integrity.
2024-06-24 | GSE226309 | GEO
Project description:Embryonic genome instability upon DNA replication timing program emergence
| PRJNA1075049 | ENA
Project description:Embryonic genome instability upon DNA replication timing program emergence
| PRJNA939700 | ENA
Project description:DNA is replicated according to a defined spatiotemporal program that is linked to both gene regulation and genome stability. The evolutionary forces that have shaped replication timing programs in eukaryotic species are largely unknown. Here, we studied the molecular causes and consequences of replication timing evolution across 94 humans, 95 chimpanzees, and 23 rhesus macaques. Replication timing differences recapitulated the species' phylogenetic tree, suggesting continuous evolution of the DNA replication timing program in primates. Hundreds of genomic regions had significant replication timing variation between humans and chimpanzees, of which 66 showed advances in replication origin firing in humans, while 57 were delayed. Genes overlapping these regions displayed correlated changes in expression levels and chromatin structure. Many human-chimpanzee variants also exhibited interindividual replication timing variation, pointing to ongoing evolution of replication timing at these loci. Association of replication timing variation with genetic variation revealed that DNA sequence evolution can explain replication timing variation between species. Taken together, DNA replication timing shows substantial and ongoing evolution in the human lineage that is driven by sequence alterations and could impact regulatory evolution at specific genomic sites.
| S-EPMC10013799 | biostudies-literature
Project description:Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.
| S-EPMC6144785 | biostudies-literature
Project description:Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.
| S-EPMC2937526 | biostudies-literature
Project description:DNA polymerase iota (Pol ι) is a Y-family lesion bypass polymerases, characterized by its remarkably low fidelity. While Pol ι is ubiquitously expressed in nearly all cell types, its function remains largely undefined. Previous studies in cellular and mouse models deficient in Pol ι have revealed no discernible phenotype. In this report, we show that Pol ι plays an important role in mitotic DNA synthesis. Cells lacking Pol ι exhibit heightened vulnerability to replication stress, increased formation of micronuclei, and accumulation of chromosomal breaks. The protein level of Pol ι is tightly regulated and restricted to G2/M phase cells via CRL7/FBXW11-mediated proteasomal degradation. The absence of Pol ι during S phase ensures that its error-prone DNA synthesis activity is excluded from interfering with normal DNA replication. These findings suggest that Pol ι function is critical in resolving DNA damage-induced replication stress prior to the completion of mitosis.
2025-07-01 | PXD065672 |
Project description:We describe the DNA replication timing programs of 14 yeast mutants with an extended S phase identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control, and dNTP synthesis and affect different parts of S phase. In 13 of the mutants, origin activation time scales with the duration of S phase. A limited number of origins become inactive in these strains, with inactive origins characterized by small replicons and distributed throughout S phase. In sharp contrast, cells deleted of MRC1, a gene implicated in replication fork stabilization and in the replication checkpoint pathway, maintained wild-type firing times despite over twofold lengthening of S phase. Numerous dormant origins were activated in this mutant. Our data suggest that most perturbations that lengthen S phase affect the entire program of replication timing, rather than a specific subset of origins, maintaining the relative order of origin firing time and delaying firing with relative proportions. Mrc1 emerges as a regulator of this robustness of the replication program.
| S-EPMC2877575 | biostudies-literature
Project description:The organization of mammalian DNA replication is poorly understood. We have produced high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal, and embryonic stem (ES) cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy-number variations during S phase to produce replication timing profiles. We first obtained timing maps of 3% of the genome using high-density oligonucleotide tiling arrays and then extended the TimEX method genome-wide using massively parallel sequencing. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation, where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by single molecule analysis of replicated DNA (SMARD). Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarters of S phase. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue-specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid-firing origins are located near moderately expressed genes, and that late-firing origins are located far from genes.
| S-EPMC2792175 | biostudies-literature