Replication Fork Velocities at Adjacent Replication Origins Are Coordinately Modified during DNA Replication in Human Cells

The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.

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Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

Author(s):

D. T. Gruszka ◽

S. Xie ◽

H. Kimura ◽

H. Yardimci

Keyword(s):

Dna Replication ◽

Real Time ◽

Single Molecule ◽

Replication Fork ◽

Epigenetic Inheritance ◽

Replication Forks ◽

Replication Fork Progression ◽

Egg Extracts ◽

Fork Stalling ◽

The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

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Helicases that interact with replication forks: new candidates from archaea

Biochemical Society Transactions ◽

10.1042/bst0331471 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1471-1473 ◽

Cited By ~ 1

Author(s):

E.L. Bolt

Keyword(s):

Cell Division ◽

Dna Replication ◽

Replication Fork ◽

Genome Duplication ◽

Genome Instability ◽

Replication Forks ◽

Valuable Resource ◽

Replication Restart

Overcoming DNA replication fork blocks is essential for completing genome duplication and cell division. Archaea and eukaryotes drive replication using essentially the same protein machinery. Archaea may be a valuable resource for identifying new helicase components at advancing forks and/or in replication-restart pathways. As described here, these may be relevant to understanding genome instability in metazoans.

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Replication Fork Slowing and Stalling are Distinct, Checkpoint-Independent Consequences of Replicating Damaged DNA

10.1101/122895 ◽

2017 ◽

Author(s):

Divya Ramalingam Iyer ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Single Molecule ◽

Replication Fork ◽

Replication Forks ◽

Origin Firing ◽

Dna Combing ◽

Fork Stalling

AbstractIn response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.Author SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kelsey Whinn ◽

Gurleen Kaur ◽

Jacob S. Lewis ◽

Grant Schauer ◽

Stefan Müller ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Fork ◽

Molecular Machines ◽

Replication Forks ◽

Chromosomal Dna ◽

Single Molecule Level ◽

Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

Nature Communications ◽

10.1038/ncomms3368 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 46

Author(s):

Maria Manosas ◽

Senthil K. Perumal ◽

Piero R. Bianco ◽

Felix Ritort ◽

Stephen J. Benkovic ◽

...

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Single Molecule ◽

Replication Fork ◽

Genetic Recombination ◽

Bacteriophage T4 ◽

General Mechanism ◽

Dna Unwinding ◽

Replication Forks ◽

Displacement Reactions

Abstract Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.

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Stochasticity of replication fork speed plays a key role in the dynamics of DNA replication

10.1101/696625 ◽

2019 ◽

Author(s):

Razie Yousefi ◽

Maga Rowicka

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Computational Models ◽

Replication Timing ◽

Temporal Organization ◽

Yeast Cells ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Timely Completion ◽

Spatio Temporal

AbstractEukaryotic DNA replication is elaborately orchestrated to duplicate the genome timely and faithfully. Replication initiates at multiple origins from which replication forks emanate and travel bi-directionally. The complex spatio-temporal regulation of DNA replication remains incompletely understood. To study it, computational models of DNA replication have been developed in S. cerevisiae. However, in spite of the experimental evidence of replication speed stochasticity, all models assumed that replication fork speed is constant or varies only with genomic coordinates. Here, we present the first model of DNA replication assuming stochastic speed of the replication fork. Utilizing data from both wild-type and hydroxyurea-treated yeast cells, we show that our model is more accurate than models assuming constant fork speed and reconstructs dynamics of DNA replication faithfully starting both from population-wide data and data reflecting fork movement in individual cells. Completion of replication in a timely manner is a challenge due to its stochasticity; we propose an empirically derived modification to replication speed based on the distance to the approaching fork, which promotes timely completion of replication. In summary, our work discovers a key role that stochasticity of the fork speed plays in the dynamics of DNA replication. We show that without including stochasticity of fork speed it is not possible to accurately reconstruct movement of individual replication forks, measured by DNA combing.Author summaryDNA replication in eukaryotes starts from multiple sites termed replication origins. Replication timing at individual sites is stochastic, but reproducible population-wide. Complex and not yet completely understood mechanisms ensure that genome is replicated exactly once and that replication is finished in time. This complex spatio-temporal organization of DNA replication makes computational modeling a useful tool to study replication mechanisms. For simplicity, all previous models assumed constant replication fork speed. Here, we show that such models are incapable of accurately reconstructing distances travelled by individual replication forks. Therefore, we propose a model with a stochastic replication fork speed. We show that such model reproduces faithfully distances travelled by individual replication forks. Moreover, our model is simpler than previous model and thus avoids over-learning (fitting noise). We also discover how replication speed may be attuned to timely complete replication. We propose that fork speed exponentially increases with diminishing distance to the approaching fork, which we show promotes timely completion of replication. Such speed up can be e.g. explained by a synergy effect of chromatin unwinding by both forks. Our model can be used to simulate phenomena beyond replication, e.g. DNA double-strand breaks resulting from broken replication forks.

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Factors affecting template switch recombination associated with restarted DNA replication

eLife ◽

10.7554/elife.41697 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 19

Author(s):

Manisha Jalan ◽

Judith Oehler ◽

Carl A Morrow ◽

Fekret Osman ◽

Matthew C Whitby

Keyword(s):

Homologous Recombination ◽

Replication Fork ◽

Genome Duplication ◽

Rna Polymerase Iii ◽

Dna Helicase ◽

Template Switching ◽

Replication Forks ◽

Factors Affecting ◽

Timely Completion ◽

Template Switch

Homologous recombination helps ensure the timely completion of genome duplication by restarting collapsed replication forks. However, this beneficial function is not without risk as replication restarted by homologous recombination is prone to template switching (TS) that can generate deleterious genome rearrangements associated with diseases such as cancer. Previously we established an assay for studying TS in Schizosaccharomyces pombe (Nguyen et al., 2015). Here, we show that TS is detected up to 75 kb downstream of a collapsed replication fork and can be triggered by head-on collision between the restarted fork and RNA Polymerase III transcription. The Pif1 DNA helicase, Pfh1, promotes efficient restart and also suppresses TS. A further three conserved helicases (Fbh1, Rqh1 and Srs2) strongly suppress TS, but there is no change in TS frequency in cells lacking Fml1 or Mus81. We discuss how these factors likely influence TS.

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Single-molecule insight into stalled replication fork rescue in Escherichia coli

Nucleic Acids Research ◽

10.1093/nar/gkab142 ◽

2021 ◽

Author(s):

Piero R Bianco ◽

Yue Lu

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Single Molecule ◽

Replication Fork ◽

Protein Complexes ◽

Replication Forks ◽

Step Process ◽

Sequential Action ◽

Single Molecule Studies ◽

Insight Into

Abstract DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

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Single-molecule binding characterization of primosomal protein PriA involved in replication restart

Physical Chemistry Chemical Physics ◽

10.1039/d1cp00638j ◽

2021 ◽

Author(s):

Tzu-Yu Lee ◽

Yi-Ching Li ◽

Min-Guan Lin ◽

Chwan-Deng Hsiao ◽

Hung-Wen Li

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Single Molecule ◽

Replication Forks ◽

Bacterial Survival ◽

Dna Damages ◽

Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

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Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

10.1101/2020.10.26.355008 ◽

2020 ◽

Author(s):

Christophe de La Roche Saint-André ◽

Vincent Géli

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genetic Material ◽

S Phase ◽

Replication Forks ◽

H3k4 Methylation ◽

Origin Firing ◽

Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

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