Transcription-dependent regulation of replication dynamics modulates genome stability

Replication stress is a primary threat to genome stability and has been implicated in tumorigenesis1, 2. Common fragile sites (CFSs) are loci hypersensitive to replication stress3 and are hotspots for chromosomal rearrangements in cancers4. CFSs replicate late in S-phase3, are cell-type dependent4–6 and nest within very large genes4, 7–9. The mechanisms responsible for CFS instability are still discussed, notably the relative impact of transcription-replication conflicts7, 8, 10versus their low density in replication initiation events5, 6. Here we address the relationships between transcription, replication, gene size and instability by manipulating the transcription of three endogenous large genes, two in chicken and one in human cells. Remarkably, moderate transcription destabilises large genes whereas high transcription levels alleviate their instability. Replication dynamics analyses showed that transcription quantitatively shapes the replication program of large genes, setting both their initiation profile and their replication timing as well as regulating internal fork velocity. Noticeably, high transcription levels advance the replication time of large genes from late to mid S-phase, which most likely gives cells more time to complete replication before mitotic entry. Transcription can therefore contribute to maintaining the integrity of some difficult-to-replicate loci, challenging the dominant view that it is exclusively a threat to genome stability.

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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.

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

Nature Communications ◽

10.1038/s41467-019-13674-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

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 upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses 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, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription–replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription–replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

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Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

10.1101/716951 ◽

2019 ◽

Cited By ~ 1

Author(s):

Dan Sarni ◽

Takayo Sasaki ◽

Karin Miron ◽

Michal Irony Tur-Sinai ◽

Juan Carlos Rivera-Mulia ◽

...

Keyword(s):

Dna Replication ◽

Chromosomal Instability ◽

Temporal Order ◽

Replication Timing ◽

Replication Stress ◽

Fragile Sites ◽

Precise Mapping ◽

Chromosomal Fragility ◽

Transcriptional Changes ◽

Large Genes

AbstractCommon fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

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Regulation of DNA Replication Licensing and Re-Replication by Cdt1

International Journal of Molecular Sciences ◽

10.3390/ijms22105195 ◽

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.

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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.

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Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.00260-17 ◽

2017 ◽

Vol 37 (22) ◽

Cited By ~ 2

Author(s):

Michael C. Reubens ◽

Sophie Rozenzhak ◽

Paul Russell

Keyword(s):

Genome Stability ◽

Genome Instability ◽

Replication Stress ◽

S Phase ◽

Dna Lesions ◽

Brct Domain ◽

Replication Forks ◽

Content Type ◽

Domain Protein ◽

Chromosome Integrity

ABSTRACT DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

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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.

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Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq

Cytogenetic and Genome Research ◽

10.1159/000518263 ◽

2021 ◽

pp. 1-8

Author(s):

Takuya Hayakawa ◽

Rino Suzuki ◽

Kazuhiro Kagotani ◽

Katsuzumi Okumura ◽

Shin-ichiro Takebayashi

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Topoisomerase I ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Replication Timing ◽

Replication Stress ◽

Replication Initiation ◽

Domain Structures ◽

Early Replication

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

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Inhibition of Human Chk1 Causes Increased Initiation of DNA Replication, Phosphorylation of ATR Targets, and DNA Breakage

Molecular and Cellular Biology ◽

10.1128/mcb.25.9.3553-3562.2005 ◽

2005 ◽

Vol 25 (9) ◽

pp. 3553-3562 ◽

Cited By ~ 357

Author(s):

Randi G. Syljuåsen ◽

Claus Storgaard Sørensen ◽

Lasse Tengbjerg Hansen ◽

Kasper Fugger ◽

Cecilia Lundin ◽

...

Keyword(s):

Dna Replication ◽

Genome Stability ◽

Protein A ◽

S Phase ◽

Replication Initiation ◽

Dna Breakage ◽

Histone H2ax ◽

Initiation Of Dna Replication ◽

Dna Strand ◽

Interfering Rna

ABSTRACT Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.

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Epstein-Barr Virus Episome Stability Is Coupled to a Delay in Replication Timing

Journal of Virology ◽

10.1128/jvi.02115-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2154-2162 ◽

Cited By ~ 31

Author(s):

Jing Zhou ◽

Andrew R. Snyder ◽

Paul M. Lieberman

Keyword(s):

Histone Deacetylases ◽

Genome Stability ◽

Epstein Barr Virus ◽

Replication Timing ◽

Replication Initiation ◽

Stable Complex ◽

Telomere Repeat ◽

Temporal Regulation ◽

Barr Virus ◽

Epstein Barr

ABSTRACT The temporal regulation of DNA replication is thought to be important for chromosome organization and genome stability. We show here that Epstein-Barr virus (EBV) genomes replicate in mid- to late S phase and that agents that accelerate replication timing of EBV reduce viral genome stability. Hydroxyurea (HU) treatment, which is known to eliminate EBV episomes, shifted EBV replication to earlier times in the cell cycle. HU treatment correlated with hyperacetylation of histone H3 and loss of telomere repeat factor 2 (TRF2) binding at the EBV origin of plasmid replication (OriP). Deletion of TRF2 binding sites within OriP or short hairpin RNA depletion of TRF2 advanced the replication timing of OriP-containing plasmids. Inhibitors of class I histone deacetylases (HDACs) increased histone acetylation at OriP, advanced the replication timing of EBV, and reduced EBV genome copy number. We also show that HDAC1 and -2 form a stable complex with TRF2 at OriP and that HU treatment inhibits HDAC activity. We propose that the TRF2-HDAC complex enhances EBV episome stability by providing a checkpoint that delays replication initiation at OriP.

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