Genome-Wide Identification of Early-Firing Human Replication Origins by Optical Replication Mapping

AbstractThe timing of DNA replication is largely regulated by the location and timing of replication origin firing. Therefore, much effort has been invested in identifying and analyzing human replication origins. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual origins in metazoans has made mapping the location and timing of replication initiation in human cells difficult. We have mapped early-firing origins in HeLa cells using Optical Replication Mapping, a high-throughput single-molecule approach based on Bionano Genomics genomic mapping technology. The single-molecule nature and 290-fold coverage of our dataset allowed us to identify origins that fire with as little as 1% efficiency. We find sites of human replication initiation in early S phase are not confined to well-defined efficient replication origins, but are instead distributed across broad initiation zones consisting of many inefficient origins. These early-firing initiation zones co-localize with initiation zones inferred from Okazaki-fragment-mapping analysis and are enriched in ORC1 binding sites. Although most early-firing origins fire in early-replication regions of the genome, a significant number fire in late-replicating regions, suggesting that the major difference between origins in early and late replicating regions is their probability of firing in early S-phase, as opposed to qualitative differences in their firing-time distributions. This observation is consistent with stochastic models of origin timing regulation, which explain the regulation of replication timing in yeast.

Download Full-text

Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing at HMRa

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1955-1966.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1955-1966 ◽

Cited By ~ 7

Author(s):

Kristopher H. McConnell ◽

Philipp Müller ◽

Catherine A. Fox

Keyword(s):

Origin Recognition Complex ◽

Substantial Reduction ◽

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Silent Chromatin ◽

Replication Origins ◽

Origin Firing ◽

Sir Proteins ◽

Origin Recognition

ABSTRACT The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMR a locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMR a. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMR a silencing, origin firing, and replication timing. Origin firing within HMR a and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMR a replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMR a quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.

Download Full-text

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

10.1101/2020.08.24.263459 ◽

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.

Download Full-text

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication

10.1101/2021.03.17.435814 ◽

2021 ◽

Author(s):

Rachel A. Hoffman ◽

David M. MacAlpine

Keyword(s):

Dna Replication ◽

S Phase ◽

Replication Initiation ◽

Micrococcal Nuclease ◽

Sequence Context ◽

Α Subunit ◽

Chromatin Dynamics ◽

Conditional Mutant ◽

Local Sequence ◽

Efficient Replication

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S-phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin which extends up to one kilobase from early, efficient replication origins. The CMG holo-helicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex, but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

Download Full-text

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

10.1101/2021.05.14.443897 ◽

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.

Download Full-text

Dynamics of DNA replication in a eukaryotic cell

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818680116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4973-4982 ◽

Cited By ~ 18

Author(s):

Thomas Kelly ◽

A. John Callegari

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Origin Recognition Complex ◽

Complex Dynamics ◽

Replication Timing ◽

Eukaryotic Cell ◽

Replication Initiation ◽

Integrative Model ◽

Genomic Locus ◽

Two Factors

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

Download Full-text

Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1

Open Biology ◽

10.1098/rsob.130138 ◽

2014 ◽

Vol 4 (1) ◽

pp. 130138 ◽

Cited By ~ 18

Author(s):

Wei Theng Poh ◽

Gaganmeet Singh Chadha ◽

Peter J. Gillespie ◽

Philipp Kaldis ◽

J. Julian Blow

Keyword(s):

Protein Phosphatase ◽

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Protein Phosphatase 1 ◽

Checkpoint Kinases ◽

Anti Cancer ◽

Egg Extracts ◽

Mcm Proteins

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.

Download Full-text

DNA Replication Origins Fire Stochastically in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0657 ◽

2006 ◽

Vol 17 (1) ◽

pp. 308-316 ◽

Cited By ~ 133

Author(s):

Prasanta K. Patel ◽

Benoit Arcangioli ◽

Stephen P. Baker ◽

Aaron Bensimon ◽

Nicholas Rhind

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Replication Initiation ◽

Epigenetic Mechanism ◽

Replication Origins ◽

Origin Firing ◽

Cell Cycles ◽

Initiation Of Replication ◽

Dna Combing ◽

Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

Download Full-text

Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0020 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4374-4382 ◽

Cited By ~ 13

Author(s):

Ling Yin ◽

Alexandra Monica Locovei ◽

Gennaro D'Urso

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

S Phase ◽

Replication Initiation ◽

Dna Damage Checkpoint ◽

Origin Firing ◽

Dna Replication Initiation ◽

Fork Stalling ◽

S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

Download Full-text

Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

10.1101/714717 ◽

2019 ◽

Cited By ~ 1

Author(s):

Olivier Brison ◽

Sami EL-Hilali ◽

Dana Azar ◽

Stéphane Koundrioukoff ◽

Mélanie Schmidt ◽

...

Keyword(s):

Replication Timing ◽

S Phase ◽

Fragile Sites ◽

Replication Initiation ◽

Common Fragile Sites ◽

Large Gene ◽

Genome Wide ◽

Underlying Mechanisms ◽

Large Genes ◽

Nascent Transcripts

ABSTRACTCommon Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

Download Full-text

Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

10.1101/2020.10.21.348946 ◽

2020 ◽

Author(s):

Emilia Puig Lombardi ◽

Madalena Tarsounas

Keyword(s):

Binding Sites ◽

Replication Fork ◽

Replication Timing ◽

Replication Initiation ◽

Ctcf Binding ◽

Origin Firing ◽

Replication Fork Progression ◽

Fork Stalling ◽

Genomic Regions ◽

The Impact

ABSTRACTTopologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

Download Full-text