scholarly journals Distinct roles of structure-specific endonucleases EEPD1 and Metnase in replication stress responses

NAR Cancer ◽  
2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Neelam Sharma ◽  
Michael C Speed ◽  
Christopher P Allen ◽  
David G Maranon ◽  
Elizabeth Williamson ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Lines ◽  
Stress Responses ◽  
Genome Instability ◽  
Replication Stress ◽  
Cancer Etiology ◽  
Double Knockout ◽  
Damage Response ◽  
Stalled Replication Forks ◽  
Single Knockout

Abstract Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.

scholarly journals Histone dynamics during DNA replication stress

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Chia-Ling Hsu ◽  
Shin Yen Chong ◽  
Chia-Yeh Lin ◽  
Cheng-Fu Kao
Keyword(s):  
Stress Responses ◽  
Genome Stability ◽  
Genome Instability ◽  
Replication Stress ◽  
Dna Lesions ◽  
Protective Mechanisms ◽  
Replication Process ◽  
Dna Replication Stress ◽  
External Sources

AbstractAccurate and complete replication of the genome is essential not only for genome stability but also for cell viability. However, cells face constant threats to the replication process, such as spontaneous DNA modifications and DNA lesions from endogenous and external sources. Any obstacle that slows down replication forks or perturbs replication dynamics is generally considered to be a form of replication stress, and the past decade has seen numerous advances in our understanding of how cells respond to and resolve such challenges. Furthermore, recent studies have also uncovered links between defects in replication stress responses and genome instability or various diseases, such as cancer. Because replication stress takes place in the context of chromatin, histone dynamics play key roles in modulating fork progression and replication stress responses. Here, we summarize the current understanding of histone dynamics in replication stress, highlighting recent advances in the characterization of fork-protective mechanisms.

scholarly journals Prevention of DNA Replication Stress by CHK1 Leads to Chemoresistance Despite a DNA Repair Defect in Homologous Recombination in Breast Cancer

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 238 ◽  
Author(s):  
Felix Meyer ◽  
Saskia Becker ◽  
Sandra Classen ◽  
Ann Christin Parplys ◽  
Wael Yassin Mansour ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Triple Negative Breast Cancer ◽  
Chromosomal Instability ◽  
Triple Negative ◽  
Replication Stress ◽  
S Phase ◽  
Damage Response ◽  
Repair Defect

Chromosomal instability not only has a negative effect on survival in triple-negative breast cancer, but also on the well treatable subgroup of luminal A tumors. This suggests a general mechanism independent of subtypes. Increased chromosomal instability (CIN) in triple-negative breast cancer (TNBC) is attributed to a defect in the DNA repair pathway homologous recombination. Homologous recombination (HR) prevents genomic instability by repair and protection of replication. It is unclear whether genetic alterations actually lead to a repair defect or whether superior signaling pathways are of greater importance. Previous studies focused exclusively on the repair function of HR. Here, we show that the regulation of HR by the intra-S-phase damage response at the replication is of overriding importance. A damage response activated by Ataxia telangiectasia and Rad3 related-checkpoint kinase 1 (ATR-CHK1) can prevent replication stress and leads to resistance formation. CHK1 thus has a preferred role over HR in preventing replication stress in TNBC. The signaling cascade ATR-CHK1 can compensate for a double-strand break repair error and lead to resistance of HR-deficient tumors. Established methods for the identification of HR-deficient tumors for Poly(ADP-Ribose)-Polymerase 1 (PARP1) inhibitor therapies should be extended to include analysis of candidates for intra-S phase damage response.

scholarly journals Treacle and TOPBP1 control replication stress response in the nucleolus

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Artem K. Velichko ◽  
Natalia Ovsyannikova ◽  
Nadezhda V. Petrova ◽  
Artem V. Luzhin ◽  
Maria Vorobjeva ◽  
...  
Keyword(s):  
Stress Response ◽  
Genome Instability ◽  
Replication Stress ◽  
Low Complexity ◽  
Drug Induced ◽  
Response Factors ◽  
Checkpoint Activation ◽  
Stalled Replication Forks

Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress–response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle–TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle–TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.

scholarly journals Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

2021 ◽  
Author(s):  
Tanay Thakar ◽  
Joshua Straka ◽  
Claudia M Nicolae ◽  
George-Lucian Moldovan
Keyword(s):  
Genome Instability ◽  
Replication Stress ◽  
Dependent Manner ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Critical Determinant ◽  
Single Stranded Dna ◽  
Histone Deposition ◽  
Stalled Replication Forks ◽  
Lagging Strand

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Polα-dependent lagging strand gaps, which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.

scholarly journals Roles of Claspin in regulation of DNA replication, replication stress responses and oncogenesis in human cells

2021 ◽  
Author(s):  
Hao-Wen Hsiao ◽  
Chi-Chun Yang ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Stress Responses ◽  
Replication Fork ◽  
Therapeutic Potential ◽  
Genome Instability ◽  
Intramolecular Interaction ◽  
Replication Stress ◽  
Human Cells ◽  
Entire Genome ◽  
Replication Checkpoint

AbstractHuman cells need to cope with the stalling of DNA replication to complete replication of the entire genome to minimize genome instability. They respond to “replication stress” by activating the conserved ATR-Claspin-Chk1 replication checkpoint pathway. The stalled replication fork is detected and stabilized by the checkpoint proteins to prevent disintegration of the replication fork, to remove the lesion or problems that are causing fork block, and to facilitate the continuation of fork progression. Claspin, a factor conserved from yeasts to human, plays a crucial role as a mediator that transmits the replication fork arrest signal from the sensor kinase, ataxia telangiectasia and Rad3-related (ATR), to the effector kinase, Checkpoint kinase 1 (Chk1). Claspin interacts with multiple kinases and replication factors and facilitates efficient replication fork progression and initiation during the normal course of DNA replication as well. It interacts with Cdc7 kinase through the acidic patch segment near the C-terminus and this interaction is critical for efficient phosphorylation of Mcm in non-cancer cells and also for checkpoint activation. Phosphorylation of Claspin by Cdc7, recruited to the acidic patch, regulates the conformation of Claspin through affecting the intramolecular interaction between the N- and C-terminal segments of Claspin. Abundance of Claspin is regulated at both mRNA and protein levels (post-transcriptional regulation and protein stability) and affects the extent of replication checkpoint. In this article, we will discuss how the ATR-Claspin-Chk1 regulates normal and stressed DNA replication and provide insight into the therapeutic potential of targeting replication checkpoint for efficient cancer cell death.

scholarly journals FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination

2020 ◽  
Vol 117 (32) ◽  
pp. 19415-19424 ◽  
Author(s):  
Elaine Guo ◽  
Yuki Ishii ◽  
James Mueller ◽  
Anjana Srivatsan ◽  
Timothy Gahman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Lines ◽  
Small Molecule ◽  
Genome Instability ◽  
Synthetic Lethality ◽  
Resistant Cell ◽  
Differential Sensitivity ◽  
Genetic Defects ◽  
Small Interfering Rnas ◽  
Synthetic Lethal Interactions

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we showRAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions withSaccharomyces cerevisiaegenome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killedBRCA1- andBRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting fromBRCA1andBRCA2mutations, and other genetic defects.

BAP1 promotes stalled fork restart and cell survival via INO80 in response to replication stress

2019 ◽  
Vol 476 (20) ◽  
pp. 3053-3066 ◽  
Author(s):  
Han-Sae Lee ◽  
Hye-Ran Seo ◽  
Shin-Ai Lee ◽  
Soohee Choi ◽  
Dongmin Kang ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Genome Stability ◽  
Genome Instability ◽  
Ectopic Expression ◽  
Replication Stress ◽  
Replication Forks ◽  
Suppressor Activity ◽  
Replication Fork Progression ◽  
Tumor Suppressor Activity ◽  
Stalled Replication Forks

Abstract The recovery from replication stress by restarting stalled forks to continue DNA synthesis is crucial for maintaining genome stability and thereby preventing diseases such as cancer. We previously showed that BRCA1-associated protein 1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, promotes replication fork progression by stabilizing the INO80 chromatin remodeler via deubiquitination and recruiting it to replication forks during normal DNA synthesis. However, whether BAP1 functions in DNA replication under stress conditions is unknown. Here, we show that BAP1 depletion reduces S-phase progression and DNA synthesis after treatment with hydroxyurea (HU). BAP1-depleted cells exhibit a defect in the restart of HU-induced stalled replication forks, which is recovered by the ectopic expression of INO80. Both BAP1 and INO80 bind chromatin at replication forks upon HU treatment. BAP1 depletion abrogates the binding of INO80 to replication forks and increases the formation of RAD51 foci following HU treatment. BAP1-depleted cells show hypersensitivity to HU treatment, which is rescued by INO80 expression. These results suggest that BAP1 promotes the restart of stress-induced stalled replication forks by recruiting INO80 to the stalled forks. This function of BAP1 in replication stress recovery may contribute to its ability to suppress genome instability and cancer development.

scholarly journals Advances in understanding DNA processing and protection at stalled replication forks

2019 ◽  
Vol 218 (4) ◽  
pp. 1096-1107 ◽  
Author(s):  
Kimberly Rickman ◽  
Agata Smogorzewska
Keyword(s):  
Mammalian Cells ◽  
Replication Stress ◽  
Genome Integrity ◽  
Replication Forks ◽  
Damage Response ◽  
Dna Translocases ◽  
Brca1 Brca2 ◽  
Stalled Replication Forks ◽  
Insight Into

The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.

scholarly journals Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

2019 ◽  
Author(s):  
Stephan B. Dreyer ◽  
Rosie Upstill-Goddard ◽  
Viola Paulus-Hock ◽  
Clara Paris ◽  
Eirini-Maria Lampraki ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Parp Inhibitor ◽  
Replication Stress ◽  
Therapeutic Strategies ◽  
Pancreatic Tumors ◽  
Damage Response

ABSTRACTContinuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.Abstract FigureSTATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

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