scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

scholarly journals Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci

1993 ◽  
Vol 13 (5) ◽  
pp. 2971-2981
Author(s):  
S M Carroll ◽  
M L DeRose ◽  
J L Kolman ◽  
G H Nonet ◽  
R E Kelly ◽  
...  
Keyword(s):  
Dna Replication ◽  
Adenosine Deaminase ◽  
Gene Amplification ◽  
Replication Origin ◽  
Replication Timing ◽  
Single Copy ◽  
S Phase ◽  
Chromosomal Locus ◽  
Template Strand ◽  
Dna Replication Origin

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.

scholarly journals Comprehensive analysis of DNA replication timing in genetic diseases and gene knockouts identifies MCM10 as a novel regulator of the replication program

2021 ◽  
Author(s):  
Madison Caballero ◽  
Tiffany Ge ◽  
Ana Rita Rebelo ◽  
Seungmae Seo ◽  
Sean Kim ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cellular Proliferation ◽  
Genetic Diseases ◽  
Replication Timing ◽  
Cell Types ◽  
Replication Initiation ◽  
Timing Variability ◽  
Gene Knockouts ◽  
Dna Replication Timing ◽  
Mutant Cells

AbstractCellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

Fish oil slows S phase progression and may cause upstream shift of DHFR replication origin ori-β in CHO cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1009-C1024 ◽  
Author(s):  
Nawfal W. Istfan ◽  
Zhi-Yi Chen ◽  
Sybille Rex
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Origin ◽  
Dna Analysis ◽  
Corn Oil ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
S Phase ◽  
Replication Initiation ◽  
Temporal Organization

Fish oils (FOs) have been noted to reduce growth and proliferation of certain tumor cells, effects usually attributed to the content of polyunsaturated fatty acids of the n–3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of FO on cell cycle kinetics of cultured Chinese hamster ovary cells. Exponentially growing cells were labeled with 5-bromo-2′-deoxyuridine (BrdU) and analyzed by flow cytometry after 5-day treatment with exogenous fat. Bivariate BrdU-DNA analysis indicated slower progression through S phase and thus longer S phase duration time in FO- but not corn oil-treated or control cells. We hypothesize that FO treatment might interfere with spatial/temporal organization of replication origins. Therefore, we mapped the well-characterized replication origin ori-β downstream of the dihydrofolate reductase gene with the nascent strand length assay. Three DNA marker segments with known positions relative to this origin were amplified by PCR. By quantitatively assessing DNA length of the fragments in all fractions containing these markers, the location of ori-β was established. In control or corn oil-treated cells, the location of ori-β was consistent with previous studies. However, in FO-treated cells, DNA replication appears to start from a new site located farther upstream from ori-β, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.

scholarly journals Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

2019 ◽  
Author(s):  
Nina Kirstein ◽  
Alexander Buschle ◽  
Xia Wu ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
...  
Keyword(s):  
Dna Replication ◽  
Temporal Order ◽  
Replication Timing ◽  
S Phase ◽  
Replication Initiation ◽  
G1 Phase ◽  
Environmental Cues ◽  
Transcriptional Program ◽  
Active Transcription ◽  
Transcription And Replication

AbstractEukaryotic replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cell’s flexibility to respond to environmental cues.

scholarly journals Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication

2020 ◽  
Author(s):  
Weitao Wang ◽  
Kyle Klein ◽  
Karel Proesmans ◽  
Hongbo Yang ◽  
Claire Marchal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Qualitative Difference ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
Early Initiation ◽  
Firing Time ◽  
Genome Wide ◽  
Low Efficiency

AbstractDNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

scholarly journals Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

1993 ◽  
Vol 13 (5) ◽  
pp. 2971-2981 ◽  
Author(s):  
S M Carroll ◽  
M L DeRose ◽  
J L Kolman ◽  
G H Nonet ◽  
R E Kelly ◽  
...  
Keyword(s):  
Dna Replication ◽  
Adenosine Deaminase ◽  
Gene Amplification ◽  
Replication Origin ◽  
Replication Timing ◽  
Single Copy ◽  
S Phase ◽  
Chromosomal Locus ◽  
Template Strand ◽  
Dna Replication Origin

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.

scholarly journals Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yongzheng Li ◽  
Boxin Xue ◽  
Mengling Zhang ◽  
Liwei Zhang ◽  
Yingping Hou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Replication Origin ◽  
High Efficiency ◽  
S Phase ◽  
Chromatin Domain ◽  
Replication Origins ◽  
Replication Efficiency ◽  
Topologically Associating Domains ◽  
Epigenetic Signatures

Abstract Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

Sign in / Sign up

Export Citation Format

Share Document