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

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.

Download Full-text

The RECQL helicase prevents replication fork collapse during replication stress

Life Science Alliance ◽

10.26508/lsa.202000668 ◽

2020 ◽

Vol 3 (10) ◽

pp. e202000668

Author(s):

Bente Benedict ◽

Marit AE van Bueren ◽

Frank PA van Gemert ◽

Cor Lieftink ◽

Sergi Guerrero Llobet ◽

...

Keyword(s):

Cancer Cells ◽

Replication Fork ◽

Critical Role ◽

Replication Stress ◽

Strand Break ◽

Replication Forks ◽

Replication Fork Progression ◽

Dna Dsbs ◽

Fork Stalling ◽

Stalled Replication Forks

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

Download Full-text

Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery

eLife ◽

10.7554/elife.41426 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 21

Author(s):

Peter Tonzi ◽

Yandong Yin ◽

Chelsea Wei Ting Lee ◽

Eli Rothenberg ◽

Tony T Huang

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Replication Fork ◽

Genome Instability ◽

Replication Stress ◽

Replication Forks ◽

Replication Fork Progression ◽

Dna Replication Stress ◽

Dependent Cell ◽

Stalled Replication Forks

DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.

Download Full-text

Mms1 and Mms22 stabilize the replisome during replication stress

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0848 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2396-2408 ◽

Cited By ~ 13

Author(s):

Jessica A. Vaisica ◽

Anastasija Baryshnikova ◽

Michael Costanzo ◽

Charles Boone ◽

Grant W. Brown

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Replication Fork ◽

E3 Ubiquitin Ligase ◽

Replication Stress ◽

Replication Forks ◽

Replication Fork Progression ◽

Binding Partners ◽

Efficient Recovery ◽

Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

Download Full-text

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

Genes & Development ◽

10.1101/gad.202978.112 ◽

2013 ◽

Vol 27 (1) ◽

pp. 74-86 ◽

Cited By ~ 16

Author(s):

J. Rodriguez ◽

T. Tsukiyama

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Chromatin Accessibility ◽

Replication Forks ◽

Replication Fork Progression

Download Full-text

Faculty Opinions recommendation of ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717973049.793469832 ◽

2013 ◽

Author(s):

Arach Goldar

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Chromatin Accessibility ◽

Replication Forks ◽

Replication Fork Progression

Download Full-text

Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

10.1101/2020.01.09.898684 ◽

2020 ◽

Author(s):

Zainab Tayeh ◽

Kim Stegmann ◽

Antonia Kleeberg ◽

Mascha Friedrich ◽

Josephine Ann Mun Yee Choo ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Replication Stress ◽

List Type ◽

Replication Forks ◽

Loop Formation ◽

Animal Cells ◽

Replication Fork Progression ◽

Primordial Dwarfism ◽

Dna Replication Stress

AbstractCentrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication.HighlightsCentrosome defects cause replication stress independent of mitosis.MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation.Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation.Graphical abstract

Download Full-text

Chl1 helicase controls replication fork progression by regulating dNTP pools

Life Science Alliance ◽

10.26508/lsa.202101153 ◽

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.

Download Full-text

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity

10.1101/544320 ◽

2019 ◽

Author(s):

Syed Moiz Ahmed ◽

Priya Dharshana Ramani ◽

Stephen Wong Qi Rong ◽

Xiaodan Zhao ◽

Roland Ivanyi-Nagy ◽

...

Keyword(s):

Cancer Cells ◽

Dna Cleavage ◽

Replication Fork ◽

Genome Stability ◽

Replication Stress ◽

Dna Supercoiling ◽

Replication Forks ◽

Chromosomal Dna ◽

Dna Breaks ◽

Hmga2 Protein

AbstractThe transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.Author SummaryDNA replication fork stability in rapidly dividing cancer cells is of utmost importance for the maintenance of genome stability and cancer cell viability. Cancer cells efficiently prevent fork collapse into lethal double strand breaks as a first line of defense during replication stress, but the corresponding protective mechanisms often remain elusive.Uncontrolled high levels of DNA supercoiling that are generally regulated by topoisomerases can cause replication stress and are major threats to fork stability. Using a multidisciplinary approach, we identified a possible regulatory mechanism of replication stress, which appears to involve mitigating the consequences of DNA topological changes by the oncofetal replication fork chaperone HMGA2.Our work provides mechanistic insights into the control of DNA damage triggered by clinically important anti-cancer drugs, which is mediated by the replication fork chaperone HMGA2. We thereby also identify HMGA2 expression as a predictive therapeutic marker, which could allow clinicians to take informed decisions to prevent tumor recurrence and improve survival.

Download Full-text

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

Scientific Reports ◽

10.1038/srep31973 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 22

Author(s):

Martin R. Gill ◽

Siti Norain Harun ◽

Swagata Halder ◽

Ramon A. Boghozian ◽

Kristijan Ramadan ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Dna Replication ◽

Cancer Cells ◽

Replication Fork ◽

Human Cancer ◽

Cell Signalling ◽

Ionising Radiation ◽

Replication Forks ◽

Replication Fork Progression

Abstract Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

Download Full-text