ING2 controls the progression of DNA replication forks to maintain genome stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

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Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa054 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3053-3070

Author(s):

Esther C Morafraile ◽

Alberto Bugallo ◽

Raquel Carreira ◽

María Fernández ◽

Cristina Martín-Castellanos ◽

...

Keyword(s):

Genome Stability ◽

Nuclease Activity ◽

S Phase ◽

Exonuclease Activity ◽

Replication Forks ◽

Post Translational Modifications ◽

Replicative Stress ◽

Stalled Replication Forks ◽

Maintain Genome Stability ◽

Specific Contribution

Abstract The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

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Nucleolar Localization and Dynamic Roles of Flap Endonuclease 1 in Ribosomal DNA Replication and Damage Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00200-08 ◽

2008 ◽

Vol 28 (13) ◽

pp. 4310-4319 ◽

Cited By ~ 40

Author(s):

Zhigang Guo ◽

Limin Qian ◽

Ren Liu ◽

Huifang Dai ◽

Mian Zhou ◽

...

Keyword(s):

Dna Replication ◽

Genome Stability ◽

Phosphorylation Site ◽

Replication Forks ◽

Repair Capacity ◽

Flap Endonuclease 1 ◽

Damage Repair ◽

Dependent Endonuclease ◽

Cross Links ◽

Cellular Compartmentalization

ABSTRACT Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser187Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser187 by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.

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Werner Syndrome Protein and DNA Replication

International Journal of Molecular Sciences ◽

10.3390/ijms19113442 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3442 ◽

Cited By ~ 11

Author(s):

Shibani Mukherjee ◽

Debapriya Sinha ◽

Souparno Bhattacharya ◽

Kalayarasan Srinivasan ◽

Salim Abdisalaam ◽

...

Keyword(s):

Dna Replication ◽

Ataxia Telangiectasia ◽

Replication Fork ◽

Genome Stability ◽

Werner Syndrome ◽

Genotoxic Stress ◽

Premature Aging ◽

Replication Forks ◽

Werner Syndrome Protein ◽

Syndrome Protein

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3′ to 5′ helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN’s contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN’s molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

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A new role of the Mus81 nuclease for replication completion after fork restart

10.1101/785501 ◽

2019 ◽

Cited By ~ 2

Author(s):

Benjamin Pardo ◽

María Moriel-Carretero ◽

Thibaud Vicat ◽

Andrés Aguilera ◽

Philippe Pasero

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Genome Stability ◽

Molecular Level ◽

Dna Supercoiling ◽

Replication Forks ◽

Dna Topoisomerase ◽

Replication Restart ◽

Protein Crosslinks

ABSTRACTImpediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway is involved in the restart of blocked replication forks. Here, we used a new method to study at the molecular level the restart of replication in response to DNA topoisomerase I poisoning by camptothecin (CPT). We show that HR-mediated restart at the global genomic level occurs by a BIR-like mechanism that requires Rad52, Rad51 and Pol32. The Mus81 endonuclease, previously proposed to cleave blocked forks, is not required for replication restart in S phase but appears to be essential to resolve fork-associated recombination intermediates in G2/M as a step necessary to complete replication. We confirmed our results using an independent system that allowed us to conclude that this mechanism is independent of the accumulation of DNA supercoiling and DNA-protein crosslinks normally caused by CPT. Thus, we describe here a specific function for Mus81 in the processing of HR-restarted forks required to complete DNA replication.

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Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

10.1101/346650 ◽

2018 ◽

Author(s):

Alexander Munden ◽

Zhan Rong ◽

Rama Gangula ◽

Simon Mallal ◽

Jared T. Nordman

Keyword(s):

Copy Number ◽

Replication Fork ◽

Genome Stability ◽

Replication Timing ◽

Physical Interaction ◽

Dependent Manner ◽

Replication Forks ◽

Dna Copy Number ◽

Replication Fork Progression ◽

Maintain Genome Stability

ABSTRACTControl of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

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TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

The Journal of Cell Biology ◽

10.1083/jcb.201506071 ◽

2015 ◽

Vol 212 (1) ◽

pp. 63-75 ◽

Cited By ~ 39

Author(s):

Saskia Hoffmann ◽

Stine Smedegaard ◽

Kyosuke Nakamura ◽

Gulnahar B. Mortuza ◽

Markus Räschle ◽

...

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Genome Stability ◽

Replication Stress ◽

Nuclear Antigen ◽

Replication Forks ◽

Chromosomal Dna ◽

Checkpoint Signaling ◽

Processivity Factor ◽

Proliferating Cell

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.

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ZNF365 promotes stalled replication forks recovery to maintain genome stability

Cell Cycle ◽

10.4161/cc.25882 ◽

2013 ◽

Vol 12 (17) ◽

pp. 2817-2828 ◽

Cited By ~ 11

Author(s):

Yuqing Zhang ◽

Eumni Park ◽

Christopher Kim ◽

Ji-hye Paik

Keyword(s):

Genome Stability ◽

Replication Forks ◽

Stalled Replication Forks ◽

Maintain Genome Stability

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SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

10.1101/2020.05.18.097154 ◽

2020 ◽

Author(s):

Julie Rageul ◽

Jennifer J. Park ◽

Ping Ping Zeng ◽

Eun-A Lee ◽

Jihyeon Yang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Essential Role ◽

Replication Stress ◽

Replication Forks ◽

Genomic Integrity ◽

Fork Protection Complex ◽

Stalled Replication Forks ◽

Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

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Mandatory coupling of zygotic transcription to DNA replication in early Drosophila embryos

10.1101/2022.01.04.474810 ◽

2022 ◽

Author(s):

Chun-Yi Cho ◽

James P. Kemp ◽

Robert J. Duronio ◽

Patrick H. O'Farrell

Keyword(s):

Dna Replication ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Cluster Formation ◽

Embryonic Cell ◽

Rna Polymerases ◽

Replication Forks ◽

Transcriptional Initiation ◽

Cell Cycles ◽

Zygotic Transcription

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesized that collision-avoidance mechanisms safeguard the onset of zygotic transcription in these cycles. To explore this hypothesis, we used real-time imaging of transcriptional events at the onset of each interphase. Endogenously tagged RNA polymerase II (RNAPII) abruptly formed clusters before nascent transcripts accumulated, indicating recruitment prior to transcriptional engagement. Injection of inhibitors of DNA replication prevented RNAPII clustering, blocked formation of foci of the pioneer factor Zelda, and largely prevented expression of transcription reporters. Knockdown of Zelda or the histone acetyltransferase CBP prevented RNAPII cluster formation except at the replication-dependent (RD) histone gene locus. We suggest a model in which the passage of replication forks allows Zelda and a distinct pathway at the RD histone locus to reconfigure chromatin to nucleate RNAPII clustering and promote transcriptional initiation. The replication dependency of these events defers initiation of transcription and ensures that RNA polymerases transcribe behind advancing replication forks. The resulting coordination of transcription and replication explains how early embryos circumvent collisions and promote genome stability.

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