DNA Replication Modulates R-loop Levels to Maintain Genome Stability

SummaryConflicts between transcription and replication are a potent source of DNA damage. The transcription machinery has the potential to aggravate such conflicts by generating co-transcriptional R-loops as an additional barrier to replication fork progression. Here, we investigate the influence of conflict orientation and R-loop formation on genome stability in human cells using a defined episomal system. This approach reveals that head-on (HO) and co-directional (CD) conflicts induce distinct DNA damage responses. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the CD orientation but promoting their formation in the HO orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings not only uncover an intrinsic function of the replisome in R-loop homeostasis, but also suggest a mechanistic basis for genome instability associated with deregulated DNA replication, which is observed in many disease states, including cancer.

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Molecular connections between circadian rhythm and genome maintenance pathways

Endocrine Related Cancer ◽

10.1530/erc-20-0372 ◽

2020 ◽

Author(s):

Arun Mouli Kolinjivadi ◽

Siao Ting Chong ◽

Joanne Ngeow

Keyword(s):

Cell Cycle ◽

Dna Repair ◽

Dna Replication ◽

Circadian Clock ◽

Genome Stability ◽

Genome Instability ◽

Model Systems ◽

Genome Maintenance ◽

Replication Fork Progression ◽

Repair Efficiency

Co-ordinated oscillation of mammalian circadian clock and cell cycle is essential for cellular and organismal homeostasis. Existing preclinical, epidemiological, molecular and biochemical evidence reveal a robust interplay between circadian clock, genome instability and cancer. Furthermore, recent investigations have demonstrated that the alterations in circadian clock perturb genome stability by modulating the cell cycle timing, altering DNA replication fork progression, influencing DNA Damage Response (DDR) and DNA repair efficiency. In this review, we examine the most recent findings from different eukaryotic model systems and discuss the functional interaction between circadian factors with key DNA replication, DDR and DNA repair genes.

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FANCM-family branchpoint translocases remove co-transcriptional R-loops

10.1101/248161 ◽

2018 ◽

Cited By ~ 1

Author(s):

Charlotte Hodson ◽

Julienne J O’Rourke ◽

Sylvie van Twest ◽

Vincent J Murphy ◽

Elyse Dunn ◽

...

Keyword(s):

Breast Cancer ◽

Rna Polymerase ◽

Fanconi Anemia ◽

Genome Stability ◽

Genome Instability ◽

Protein A ◽

Loop Formation ◽

Biochemical Activity ◽

Mechanistic Basis

AbstractCo-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leading to formation of stable DNA:RNA hybrids. Unresolved R-loops can promote genome instability. Here, we show that the Fanconi anemia- and breast cancer-associated FANCM protein can directly unwind DNA-RNA hybrids from co-transcriptional R-loops in vitro. FANCM processively unwinds both short and long R-loops, irrespective of sequence, topology or coating by replication protein A. R-loops can also be unwound in the same assay by the yeast and bacterial orthologs of FANCM, Mph1 and RecG, indicating an evolutionary conserved function. Consistent with this biochemical activity of FANCM, we show that FANCM deficient cells are sensitive to drugs that stabilize R-loop formation. Our work reveals a mechanistic basis for R-loop metabolism that is critical for genome stability.

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Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage

The Journal of Cell Biology ◽

10.1083/jcb.202002175 ◽

2020 ◽

Vol 219 (8) ◽

Cited By ~ 5

Author(s):

Maria Chiara Raso ◽

Nikola Djoric ◽

Franziska Walser ◽

Sandra Hess ◽

Fabian Marc Schmid ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Dna Helicase ◽

Replication Forks ◽

Chromosomal Breakage ◽

Replication Fork Progression ◽

Ubiquitin System ◽

Interferon Stimulated Gene 15

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-β, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

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TDP-43 dysfunction results in R-loop accumulation and DNA replication defects

Journal of Cell Science ◽

10.1242/jcs.244129 ◽

2020 ◽

Vol 133 (20) ◽

pp. jcs244129 ◽

Cited By ~ 1

Author(s):

Matthew Wood ◽

Annabel Quinet ◽

Yea-Lih Lin ◽

Albert A. Davis ◽

Philippe Pasero ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Self Assembly ◽

Rna Binding ◽

Genome Instability ◽

Binding Protein ◽

Dependent Manner ◽

Nucleic Acid Binding ◽

Loop Formation ◽

Dna Replication Stress

ABSTRACTTAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid-binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also found that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are crucial in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.This article has an associated First Person interview with the first author of the paper.

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Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

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

Nature Communications ◽

10.1038/s41467-021-25215-0 ◽

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.

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AMPK blocks chromatin activation and consequent R-loop formation to protect genome stability following acute starvation

10.21203/rs.3.rs-378687/v1 ◽

2021 ◽

Author(s):

Bing Sun ◽

McLean Sherrin ◽

Richard Roy

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Germ Line ◽

Critical Role ◽

Significant Proportion ◽

Loop Formation ◽

C Elegans ◽

Chromatin Activation ◽

Acute Starvation

Abstract During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerably more RAD-51 foci at sites of R-loop formation, potentially sequestering it from its critical role at meiotic breaks and/or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation, where in its absence R-loops accumulate, resulting in reproductive compromise and DNA damage hypersensitivity.

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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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