scholarly journals PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

2018 ◽  
Author(s):  
Emily M. Schleicher ◽  
Adri M. Galvan ◽  
George-Lucian Moldovan ◽  
Claudia M. Nicolae
Keyword(s):  
Replication Fork ◽  
Cellular Proliferation ◽  
Gene Knockout ◽  
Replication Stress ◽  
Cellular Transformation ◽  
Tumor Formation ◽  
Dna Lesions ◽  
Lesion Bypass ◽  
Replication Fork Arrest ◽  
Adp Ribosyltransferase

ABSTRACTDuring carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and other difficult to replicate regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein PCNA and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation and replication fork elongation. Mechanistically, PARP10 overexpression alleviated cellular sensitivity to replication stress by fostering the restart of stalled replication forks. Importantly, mouse xenograft studies indicated that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation by alleviating replication stress, and suggest that targeting PARP10 may represent a novel therapeutic approach.

scholarly journals Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251188
Author(s):  
Wei-chung Tsao ◽  
Raquel Buj ◽  
Katherine M. Aird ◽  
Julia M. Sidorova ◽  
Kristin A. Eckert
Keyword(s):  
Replication Fork ◽  
Cell Model ◽  
Dna Polymerases ◽  
Replication Stress ◽  
Cellular Transformation ◽  
Dna Lesions ◽  
Human Cells ◽  
Cell Cycle Checkpoint ◽  
Drug Induced ◽  
Oncogene Activation

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS G12V overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS G12V overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol α catalytic subunit (POLA1), Pol δ catalytic and p68 subunits (POLD1 and POLD3), Pol η, and Pol κ. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol η (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol α expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-RasG12V-induced polymerase depletion, as compared to Pol η-deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.

scholarly journals Monoubiquitylation of histone H2B contributes to the bypass of DNA damage during and after DNA replication

2017 ◽  
Vol 114 (11) ◽  
pp. E2205-E2214 ◽  
Author(s):  
Shih-Hsun Hung ◽  
Ronald P. Wong ◽  
Helle D. Ulrich ◽  
Cheng-Fu Kao
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Dna Lesions ◽  
Template Switching ◽  
Lesion Bypass ◽  
Dna Damage Tolerance ◽  
Cytological Evidence ◽  
Histone H2b ◽  
Chromatin Dynamics ◽  
Dna Lesion

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18. However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

scholarly journals SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 2010
Author(s):  
Kamila Schirmeisen ◽  
Sarah A. E. Lambert ◽  
Karol Kramarz
Keyword(s):  
Homologous Recombination ◽  
Replication Fork ◽  
Replication Stress ◽  
Dna Lesions ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Replication Forks ◽  
Nuclear Positioning ◽  
Nuclear Compartment ◽  
Pore Complexes

DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

scholarly journals MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianning Lai ◽  
Ronan Broderick ◽  
Valérie Bergoglio ◽  
Jutta Zimmer ◽  
Sophie Badie ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
Replication Stress ◽  
Nuclease Activity ◽  
Dna Lesions ◽  
Genome Replication ◽  
Replication Fork Progression ◽  
Nucleolytic Activity ◽  
Genomic Regions

AbstractFailure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

scholarly journals The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

2019 ◽  
Author(s):  
Magdalena Ganz ◽  
Christopher Vogel ◽  
Christina Czada ◽  
Vera Jörke ◽  
Rebecca Kleiner ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Replication Fork ◽  
Replication Stress ◽  
Tumor Formation ◽  
Replication Forks ◽  
Functional Link ◽  
Protein Levels ◽  
Replication Fork Progression ◽  
Dna Replication Stress ◽  
Architectural Protein

ABSTRACTDNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

scholarly journals Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Venkateswarlu Gangavarapu ◽  
Sergio R. Santa Maria ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Replication Fork ◽  
Dna Lesions ◽  
Yeast Cells ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Site ◽  
Lesion Bypass ◽  
Content Type ◽  
Replication Checkpoint ◽  
Template Strand

ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.

scholarly journals Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in replication fork stability controlled by ATR

2020 ◽  
Author(s):  
Ashna Dhoonmoon ◽  
Emily M. Schleicher ◽  
Claudia M. Nicolae ◽  
Kristen E. Clements ◽  
George-Lucian Moldovan
Keyword(s):  
Replication Fork ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Replication Stress ◽  
Multiple Roles ◽  
Cycle Progression ◽  
Genetic Components ◽  
Genome Wide ◽  
The Face ◽  
Adp Ribosyltransferase

AbstractThe DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of replication fork stability as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.

scholarly journals Chl1 helicase controls replication fork progression by regulating dNTP pools

2022 ◽  
Vol 5 (4) ◽  
pp. e202101153
Author(s):  
Amandine Batté ◽  
Sophie C van der Horst ◽  
Mireille Tittel-Elmer ◽  
Su Ming Sun ◽  
Sushma Sharma ◽  
...  
Keyword(s):  
Stress Response ◽  
Replication Fork ◽  
Replication Stress ◽  
Stress Conditions ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Replication Fork Progression ◽  
Intracellular Dntp ◽  
Replication Fork Arrest

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

scholarly journals Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant

2020 ◽  
Vol 40 (14) ◽  
Author(s):  
Seong Min Kim ◽  
Susan L. Forsburg
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Genome Instability ◽  
Protein A ◽  
Replication Stress ◽  
Temperature Sensitive ◽  
Replication Checkpoint ◽  
Cycle Arrest ◽  
A Subunit ◽  
Replication Fork Arrest

ABSTRACT Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

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