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

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.

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

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
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.

scholarly journals Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

2010 ◽  
Vol 191 (7) ◽  
pp. 1285-1297 ◽  
Author(s):  
Xin Quan Ge ◽  
J. Julian Blow
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Apparent Contrast ◽  
Origin Firing ◽  
Checkpoint Kinases ◽  
Replicative Stress ◽  
Replication Fork Progression ◽  
Replication Factory ◽  
Low Levels ◽  
Fork Stalling

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

scholarly journals Transcription drives DNA replication initiation and termination in human cells

2018 ◽  
Author(s):  
Yu-Hung Chen ◽  
Sarah Keegan ◽  
Malik Kahli ◽  
Peter Tonzi ◽  
David Fenyö ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Replication Fork ◽  
Human Cells ◽  
Replication Initiation ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Origin Activation ◽  
Dna Replication Origins ◽  
The Impact

ABSTRACTThe locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

scholarly journals Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

2010 ◽  
Vol 30 (6) ◽  
pp. 1382-1396 ◽  
Author(s):  
Saravanabhavan Thangavel ◽  
Ramiro Mendoza-Maldonado ◽  
Erika Tissino ◽  
Julia M. Sidorova ◽  
Jinhu Yin ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Recq Helicases ◽  
Dna Replication Initiation ◽  
Replication Fork Progression ◽  
A Cell

ABSTRACT Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G1, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.

scholarly journals A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rebecca A. Dagg ◽  
Gijs Zonderland ◽  
Emilia Puig Lombardi ◽  
Giacomo G. Rossetti ◽  
Florian J. Groelly ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
High Throughput Sequencing ◽  
Germline Mutations ◽  
Cdk Inhibitor ◽  
Rna Seq ◽  
Gene Encoding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Transcriptional Alterations

AbstractBRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

scholarly journals Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

2008 ◽  
Vol 191 (2) ◽  
pp. 486-493 ◽  
Author(s):  
Adam M. Breier ◽  
Alan D. Grossman
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Initiation ◽  
Pass Through ◽  
Bacillus Subtilis Chromosome ◽  
Rapid Signaling ◽  
Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

scholarly journals CTCF and cohesin promote focal detachment of DNA from the nuclear lamina

2021 ◽  
Author(s):  
Tom van Schaik ◽  
Ning Qing Liu ◽  
Stefano G. Manzo ◽  
Daan Peric-Hupkes ◽  
Elzo de Wit ◽  
...  
Keyword(s):  
Stem Cells ◽  
Histone Modifications ◽  
Binding Sites ◽  
Embryonic Stem ◽  
Nuclear Lamina ◽  
Fine Tuning ◽  
Ctcf Binding ◽  
Strongly Correlated ◽  
Sharp Transition ◽  
Genomic Regions

Lamina associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina (NL). It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position a subset of LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin to invade into the LAD. To test this, we mapped genome - NL interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics. CTCF and cohesin contribute to a sharp transition in NL interactions at LAD borders, whilst LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the NL and enriched for accessible DNA and active histone modifications. Remarkably, even though NL positioning is strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization (depletion of the unloading factor WAPL) quantitatively affect NL interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on NL interactions. Combined, these findings illustrate that CTCF and cohesin do not shape LAD patterns. Rather, these proteins mediate fine-tuning of NL interactions.

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

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
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.

scholarly journals Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Genetics ◽  
2021 ◽  
Author(s):  
Souradip Das ◽  
Madison Caballero ◽  
Tatyana Kolesnikova ◽  
Igor Zhimulev ◽  
Amnon Koren ◽  
...  
Keyword(s):  
Dna Replication ◽  
Copy Number ◽  
Genome Stability ◽  
Timing Analysis ◽  
Replication Timing ◽  
Critical Function ◽  
Replication Fork Progression ◽  
Polyploid Cells ◽  
Genomic Regions ◽  
Insight Into

Abstract Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

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

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.

Sign in / Sign up

Export Citation Format

Share Document