Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these "replication timing quantitative trait loci" (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences.

Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1-HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression.

DNA replication is initiated at replication origins on chromosomes at their scheduled time during S phase of the cell cycle. Replication timing control is highly conserved among eukaryotes but the underlying mechanisms are not fully understood. Recent studies have revealed that some telomere-binding proteins regulate replication timing at late-replicating origins throughout the genome. To investigate the molecular basis of this process, we analyzed the effects of excessive elongation of telomere DNA on replication timing by deleting telomere-associated shelterin proteins in Schizosaccharomyces pombe. We found that rap1∆ and poz1∆ cells showed abnormally accelerated replication at internal late origins but not at subtelomere regions. These defects were suppressed by removal of telomere DNA and by deletion of the telomere-binding protein Taz1. Furthermore, Sds21-a counter protein phosphatase against Dbf4-dependent kinase (DDK)-accumulated at elongated telomeres in a Taz1-dependent manner but was depleted at internal late origins, indicating that highly elongated telomeres sequester Sds21 at telomeres and perturb replication timing at internal regions. These results demonstrate that telomere DNA length is an important determinant of replication timing at internal regions of chromosomes in eukaryotes.

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.

Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

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.

Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis. FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of double-strand break-associated factors, including 53BP1 and BRCA1. FUS-/- cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of prereplication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase-associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS complementary DNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

DNA replication plays an important role in mutagenesis, yet little is known about how it interacts with other mutagenic processes. Here, we use somatic mutation signatures-each representing a mutagenic process-derived from 3056 patients spanning 19 cancer types to quantify the strand asymmetry of mutational signatures around replication origins and between early and late replicating regions.

Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.

Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.

Induced pluripotent stem cells (iPSC) are a widely used cell system and a foundation for cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which have the potential to affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing - a process linked to both genome regulation and genome stability - is efficiently reprogrammed to the embryonic state. To answer this, we profiled and compared genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicated their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibit delayed replication at heterochromatic regions containing genes downregulated in iPSC with incompletely reprogrammed DNA methylation. DNA replication delays were not the result of gene expression and DNA methylation aberrations and persisted after differentiating cells to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and lead to undesirable phenotypes in iPSCs, establishing it as an important genomic feature to consider when evaluating iPSC lines.

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.

During the S-phase of eukaryotic cell cycle, DNA is replicated in a dedicatedly regulated temporal order, with regions containing active and inactive genes replicated early and late, respectively. Recent advances in sequencing technology allow us to explore the connection between replication timing (RT), histone modifications, and three-dimensional (3D) chromatin structure in diverse cell types. To characterize the dynamics during cell differentiation, corresponding sequencing data for human embryonic stem cells and four differentiated cell types were collected. By comparing RT and its extent of conservation before and after germ layer specification, the human genome was partitioned into distinct categories. Each category is then subject to comparisons on genomic, epigenetic, and chromatin 3D structural features. As expected, while constitutive early and late replication regions showed active and inactive features, respectively, dynamic regions with switched RT showed intermediate features. Surprisingly, although early-to-late replication and late-to-early replication regions showed similar histone modification patterns in hESCs, their structural preferences were opposite. Specifically, in hESCs, early-to-late replication regions tended to appear in the B compartment and large topologically associated domains, while late-to-early replication regions showed the opposite. Our results uncover the coordinated regulation of RT and 3D genome structure that underlies the loss of pluripotency and lineage commitment and indicate the importance and potential roles of genome architecture in biological processes.

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

In dividing cells, DNA replication occurs in a precise order, but many questions remain regarding the mechanisms of replication timing establishment and regulation. We now have generated genome-wide, high-resolution replication timing maps throughout zebrafish development. Unexpectedly, in the rapid cell cycles preceding the midblastula transition, a defined timing program was present that predicted the initial wave of zygotic transcription. Replication timing was thereafter progressively and continuously remodeled across the majority of the genome, and epigenetic changes involved in enhancer activation frequently paralleled developmental changes in replication timing. The long arm of Chromosome 4 underwent a dramatic developmentally regulated switch to late replication during gastrulation, reminiscent of mammalian X Chromosome inactivation. This study reveals that replication timing is dynamic and tightly linked to epigenetic and transcriptional changes throughout early zebrafish development. These data provide insight into the regulation and functions of replication timing and will enable further mechanistic studies.

In metazoans, each cell type follows a characteristic, spatio-temporally regulated DNA replication program. Histone modifications (HMs) and chromatin binding proteins (CBPs) are fundamental for a faithful progression and completion of this process. However, no individual HM is strictly indispensable for origin function, suggesting that HMs may act combinatorially in analogy to the histone code hypothesis for transcriptional regulation. In contrast to gene expression however, the relationship between combinations of chromatin features and DNA replication timing has not yet been demonstrated. Here, by exploiting a comprehensive data collection consisting of 95 CBPs and HMs we investigated their combinatorial potential for the prediction of DNA replication timing in Drosophila using quantitative statistical models. We found that while combinations of CBPs exhibit moderate predictive power for replication timing, pairwise interactions between HMs lead to accurate predictions genome-wide that can be locally further improved by CBPs. Independent feature importance and model analyses led us to derive a simplified, biologically interpretable model of the relationship between chromatin landscape and replication timing reaching 80% of the full model accuracy using six model terms. Finally, we show that pairwise combinations of HMs are able to predict differential DNA replication timing across different cell types. All in all, our work provides support to the existence of combinatorial HM patterns for DNA replication and reveal cell-type independent key elements thereof, whose experimental investigation might contribute to elucidate the regulatory mode of this fundamental cellular process.

Genomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.