Search

Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 impaired protein function to a similar extent as truncating variants, and 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group (OR 2,23; 95% CI 1,62-3,07; pG/p.D162G, was also identified, which co-segregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer., Cancer Research, 82 (4), ISSN:0008-5472, ISSN:1538-7445

ATM serine/threonine kinase (ATM; previously known as ataxia‐telangiectasia mutated) plays a critical role in maintaining genomic stability and regulates multiple downstream pathways, such as DNA repair, cell cycle arrest, and apoptosis. As a serine/threonine kinase, ATM has an array of downstream phosphorylation substrates, including checkpoint effector checkpoint kinase 2 (CHK2). ATM inhibits cell cycle progression by phosphorylating and activating CHK2, which plays an important role in the formation and development of tumors and participates in DNA repair responses after double‐stranded DNA breaks. In this study, we used a recently developed mammalian functional genetic screening system to explore a series of ATM substrates and their role in DNA damage to enhance our understanding of the DNA damage response. Ubiquilin 4 (UBQLN4), which belongs to the ubiquilin family characterized by its ubiquitin‐like (UBL) and ubiquitin‐associated (UBA) domains, was identified as a new substrate for ATM. UBQLN4 is involved in various intracellular processes, such as autophagosome maturation, p21 regulation, and motor axon morphogenesis. However, the biological function of UBQLN4 remains to be elucidated. In this study, we not only identified UBQLN4 as a substrate for ATM, but also found that UBQLN4 interacts with and stabilizes the anti‐apoptotic proteins Bcl‐2‐related protein A1 (BCL2A1) and Bcl‐2‐like protein 10 (BCL2L10) and prevents mesothelioma cell apoptosis in response to DNA damage. These findings expand our understanding of the role of UBQLN4 in mesothelioma and provide new insights into potential mesothelioma treatments targeting substrates for ATM., ATM phosphorylates an array of substrates that play essential roles in DNA damage response. In this study, we used a functional genetic screening system to explore ATM substrates and identified ubiquilin 4 (UBQLN4) as a new substrate for ATM. Phosphorylated UBQLN4 prevented cytochrome c release and inhibited apoptosis in mesothelioma during DNA damage by stabilizing BCL2A1 and BCL2L10. These findings expand our understanding of the role of UBQLN4 in mesothelioma and provide a new insight into mesothelioma treatment by targeting ATM substrates.

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.

The weightlessness or microgravity, a physical factor in space, may adversely affect the health of the space travellers or astronauts. The knowledge about the effect of microgravity on human cancer cells is very limited and poorly understood. Here, we employed rotary cell culture system (RCCS) to induce simulated microgravity (SMG) and examined its effects on human promyelocytic leukemic HL-60 cells. These cells were grown in normal gravity condition (1g) for control purpose. The 72 h exposure of cells to SMG decreased cell proliferation and viability which were accompanied by the reduced expression of PCNA and phosphorylated ERK1/2 and AKT proteins. SMG increased the DNA damage as well as the expression of DNA damage sensing proteins including ATM, ATR, Chk1, Chk2 and γH2A.X. The expression of AP1, XRCC1 and APEX1 regulating BER, XPC regulating NER and MLH1 and PMS2 regulating MMR were downregulated. However, SMG increased the expression of Ku70/80, DNA-PK and Rad51, regulating NHEJ and HR. SMG induced apoptosis and increased the levels of cleaved-poly-(ADP-ribose) polymerase and cleaved-caspase-3. An increase in Bax/Bcl-2 ratio and dissipation of mitochondrial membrane potential were also observed. SMG enhanced reactive oxygen species (ROS) formation which led to the enhanced DNA damage and apoptotic cell death. Overall, SMG induced ROS, DNA damage and differential expression of DNA repair genes, and altered the overall DNA repair capacity which may activate ATM/ATR-Chk1/2 and Ku70/80 and DNA-PK-mediated apoptotic cell death.

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. TheS. cerevisiaeRad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions.

The review covers recent research on cancer as a genetic disease manifesting both sporadically and in germline through variant genomic mutations or DNA rearrangements. This change can be point mutations, chromosomal aberrations or hypermethylation leading to DNA repair failures. Defects in tumour suppressor genes (BRCA1, BRCA2, CHEK2, PTCH1, etc.) underly hereditary predisposition to breast cancer (BC) and ovarian cancer (OC) due to genome instability. Studying somatic mutations is key to the understanding of carcinogenesis mechanisms and finding apt therapies. Heterogeneity of cancers renders the tumour mutation profiling uneasy. The treatment choice and efficacy in BC and OC depends on homologous recombination defects in tumour cells usually imposed by damaged BRCA1/2 genes. CHEK2- associated neoplasms account for most hereditary BCs linked to flaws in the DNA repair machinery. Overexpression of the PTCH1 protein is the target in breast, lung, ovarian, colonic cancers, etc.Genetic research has fundamentally altered our understanding of the aetiology and pathogenesis of human malignancy. The molecular cancer phenotype is of paramount importance in the disease prognosis and treatment personalisation.

Genes involved in the homologous recombination repair pathway—as exemplified by BRCA1, BRCA2, PALB2, ATM, and CHEK2—are frequently associated with hereditary breast and ovarian cancer syndrome. Germline mutations in the loci of these genes with loss of heterozygosity or additional somatic truncation at the WT allele lead to the development of breast cancers with characteristic clinicopathological features and prominent genomic features of homologous recombination deficiency, otherwise referred to as “BRCAness.” Although clinical genetic testing for these and other genes has increased the chances of identifying pathogenic variants, there has also been an increase in the prevalence of variants of uncertain significance, which poses a challenge to patient care because of the difficulties associated with making further clinical decisions. To overcome this challenge, we sought to develop a methodology to reclassify the pathogenicity of these unknown variants using statistical modeling of BRCAness. The model was developed with Lasso logistic regression by comparing 116 genomic attributes derived from 37 BRCA1/2 biallelic mutant and 32 homologous recombination‐quiescent breast cancer exomes. The model showed 95.8% and 86.7% accuracies in the training cohort and The Cancer Genome Atlas validation cohort, respectively. Through application of the model for variant reclassification of homologous recombination‐associated hereditary breast and ovarian cancer causal genes and further assessment with clinicopathological features, we finally identified one likely pathogenic and five likely benign variants. As such, the BRCAness model developed from the tumor exome was robust and provided a reasonable basis for variant reclassification., In this study, we describe a new method for assessing the pathogenicity of variants of uncertain significance (VUS) in homologous recombination (HR)‐associated genes using the tumor phenotype (BRCAness). We developed a robust statistical model to estimate BRCAness in a breast cancer sample, derived from 116 genomic variables and obtained through whole‐exome sequencing. By applying this model for the reclassification of VUS of HR‐associated hereditary breast and ovarian cancer causal genes, we identified one likely pathogenic and five likely benign variants.

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms.DesignClinical HCC samples were collected to assess the relationship between RTN3 expression and patients’ outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo.ResultsWe found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation.ConclusionThe findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.

Extragonadal germ cell tumors (GCTs) are thought to arise as a result of local transformation of primordial gonadal cells (PGCs) that become misplaced during embryogenesis. With the exception of bilateral testis tumors, metachronous GCT (i.e., occurring at a site classically described for primary GCTs) are rare events.The clinical, radiological, and molecular data (if available) of patients with metachronous GCT were analyzed.Three Caucasian males were identified: case 1 presented with a pineal germinoma 19 years after a mediastinal seminoma that had been treated with chemotherapy, case 2 presented with a pineal non-seminomatous GCT (NSGCT) that occurred three years after a mediastinal seminoma treated with chemotherapy, and case 3 presented with a mediastinal seminoma concomitant with a suprasellar germinoma that occurred two years after a stage I testicular NSGCT treated exclusively with surgery. None of these patients had a positive family history or disorder of sex development. Molecular data were available for cases 2 and 3. In case 2, aThese rare cases should alert clinicians of the possibility of multiple GCTs that should not be considered to be relapses. The underlying physiopathology is unknown, but multiple PGC mismigrations is a likely cause. Initial treatment with cisplatin may select chemo-resistant clones, thereby making the subsequent treatment more of a challenge.

ARID1A, a component of the chromatin-remodeling complex SWI/SNF, is one of the most frequently mutated genes in human cancer. We sought to develop rational combination therapy to potentiate the efficacy of immune checkpoint blockade in ARID1A-deficient tumors. In a proteomic analysis of a data set from The Cancer Genomic Atlas, we found enhanced expression of Chk2, a DNA damage checkpoint kinase, in ARID1A-mutated/deficient tumors. Surprisingly, we found that ARID1A targets the nonchromatin substrate Chk2 for ubiquitination. Loss of ARID1A increased the Chk2 level through modulating autoubiquitination of the E3-ligase RNF8 and thereby reducing RNF8-mediated Chk2 degradation. Inhibition of the ATM/Chk2 DNA damage checkpoint axis led to replication stress and accumulation of cytosolic DNA, which subsequently activated the DNA sensor STING-mediated innate immune response in ARID1A-deficient tumors. As expected, tumors with mutation or low expression of both ARID1A and ATM/Chk2 exhibited increased tumor-infiltrating lymphocytes and were associated with longer patient survival. Notably, an ATM inhibitor selectively potentiated the efficacy of immune checkpoint blockade in ARID1A-depleted tumors but not in WT tumors. Together, these results suggest that ARID1A’s targeting of the nonchromatin substrate Chk2 for ubiquitination makes it possible to selectively modulate cancer cell–intrinsic innate immunity to enhance the antitumor activity of immune checkpoint blockade.

Background Globally, colorectal cancer (CRC) affects more than 1 million people each year. In addition to non-modifiable and other environmental risk factors, Fusobacterium nucleatum infection has been linked to CRC recently. In this study, we explored mechanisms underlying the role of Fusobacterium nucleatum infection in the progression of CRC in a mouse model. Methods C57BL/6 J-Adenomatous polyposis coli (APC) Min/J mice [APC (Min/+)] were treated with Fusobacterium nucleatum (109 cfu/mL, 0.2 mL/time/day, i.g., 12 weeks), saline, or FadA knockout (FadA−/−) Fusobacterium nucleatum. The number, size, and weight of CRC tumors were determined in isolated tumor masses. The human CRC cell lines HCT29 and HT116 were treated with lentiviral vectors overexpressing chk2 or silencing β-catenin. DNA damage was determined by Comet assay and γH2AX immunofluorescence assay and flow cytometry. The mRNA expression of chk2 was determined by RT-qPCR. Protein expression of FadA, E-cadherin, β-catenin, and chk2 were determined by Western blot analysis. Results Fusobacterium nucleatum treatment promoted DNA damage in CRC in APC (Min/+) mice. Fusobacterium nucleatum also increased the number of CRC cells that were in the S phase of the cell cycle. FadA−/− reduced tumor number, size, and burden in vivo. FadA−/− also reduced DNA damage, cell proliferation, expression of E-cadherin and chk2, and cells in the S phase. Chk2 overexpression elevated DNA damage and tumor growth in APC (Min/+) mice. Conclusions In conclusion, this study provided evidence that Fusobacterium nucleatum induced DNA damage and cell growth in CRC through FadA-dependent activation of the E-cadherin/β-catenin pathway, leading to up-regulation of chk2.

Introduction Laryngeal squamous cell carcinoma (LSCC) is the most common histological subtype of laryngeal cancer. The involved molecular mechanisms and suitable therapeutic targets for LSCC still need to be further investigated. Checkpoint kinase 2 (CHK2) participates in several cellular physiology pathways and plays a role in tumor progression. However, the roles of CHK2 in LSCC remain unclear. Methods mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) database, and bioinformatic analysis was performed. Western blot and immunohistochemical analyses were conducted to detect protein expression. MTS assays were performed to examine cell growth of LSCC-derived cell lines. Results In the present study, we found that both active form of CHK2 and total CHK2 protein expressions were up-regulated in LSCC tissues. Positive expression of CHK2 was closely associated with advanced clinical features and poor prognosis. Moreover, potential CHK2-involving bioprocesses and signaling pathways were analyzed. In addition, repressed proliferation of LSCC cells was induced by CHK2 inhibitor. Discussion Taken together, our findings elucidated that CHK2 may act as an oncogenic factor in LSCC, suggesting a potential target for clinical treatment.

Elevated levels of replicative stress in gynecological cancers arising from uncontrolled oncogenic activation, loss of key tumor suppressors, and frequent defects in the DNA repair machinery are an intrinsic vulnerability for therapeutic exploitation. The presence of replication stress activates the DNA damage response and downstream checkpoint proteins including ataxia telangiectasia and Rad3 related kinase (ATR), checkpoint kinase 1 (CHK1), and WEE1-like protein kinase (WEE1), which trigger cell cycle arrest while protecting and restoring stalled replication forks. Strategies that increase replicative stress while lowering cell cycle checkpoint thresholds may allow unrepaired DNA damage to be inappropriately carried forward in replicating cells, leading to mitotic catastrophe and cell death. Moreover, the identification of fork protection as a key mechanism of resistance to chemo- and poly (ADP-ribose) polymerase inhibitor therapy in ovarian cancer further increases the priority that should be accorded to the development of strategies targeting replicative stress. Small molecule inhibitors designed to target the DNA damage sensors, such as inhibitors of ataxia telangiectasia-mutated (ATM), ATR, CHK1 and WEE1, impair smooth cell cycle modulation and disrupt efficient DNA repair, or a combination of the above, have demonstrated interesting monotherapy and combinatorial activity, including the potential to reverse drug resistance and have entered developmental pipelines. Yet unresolved challenges lie in balancing the toxicity profile of these drugs in order to achieve a suitable therapeutic index while maintaining clinical efficacy, and selective biomarkers are urgently required. Here we describe the premise for targeting of replicative stress in gynecological cancers and discuss the clinical advancement of this strategy.

Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks, which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA double-strand breaks, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA double-strand breaks, in the budding yeast model system and in mammals.

The quality of the early embryo is critical for embryonic development and implantation. Errors during cleavage lead to aneuploidy in embryos. As a cell cycle checkpoint protein, CHK2 participates in DNA replication, cell cycle arrest and spindle assembly. However, the functions of CHK2 in early development of the mouse embryo remain largely unknown. In this study, we show that CHK2 is localized on the spindle in metaphase and mainly accumulates at spindle poles in anaphase/telophase during the first cleavage of the mouse embryo. CHK2 inhibition led to cleavage failure in early embryonic development, accompanied by abnormal spindle assembly and misaligned chromosomes. Moreover, the loss of CHK2 activity increased the level of cellular DNA damage, which resulted in oxidative stress. Then, apoptosis and autophagy were found to be active in these embryos. In summary, our results suggest that CHK2 is an essential regulator of spindle assembly and DNA repair during early embryonic development in mice.

Unrepaired DNA damage in mouse oocytes leads to apoptosis, in part via CHK2 signaling to TRP53 and TRP63. Here, Rinaldi, Bloom, and Schimenti provide evidence that CHK1 can also be involved, especially... Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g., Trip13Gt mutants), but also Spo11−/− oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all Spo11−/− and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1′s role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.

Cells organize themselves to maintain proper shape, structure, and size during growth and division for their cellular functions. However, how these cellular organizations coordinate with the cell cycle is not well understood. This review focuses on cell morphogenesis and size of the membrane-bound nucleus in the fission yeast Schizosaccharomyces pombe. Growth polarity, an important factor for cell morphogenesis, in rod-shaped fission yeast is restricted to the cell tips and dynamically changes depending on the cell cycle stage. Furthermore, nuclear size in fission yeast is proportional to the cell size, resulting in a constant ratio between nuclear volume and cellular volume (N/C ratio). This review summarizes the signaling pathway(s) involved in growth polarity control and key factors involved in N/C ratio control and provides their roles in coordination between cell organization and the cell cycle.

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol e, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase–polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint. In vitro assays using a fully reconstituted DNA replication system reveal that the checkpoint kinase Rad53 restrains CMG helicase activity to prevent DNA unwinding and collapse of stalled forks in response to replication stress.

Fission yeast Cds1 is responsible for the replication checkpoint activation and helps to protect replication fork collapse in response to hydroxyurea (HU). Here, we investigated the role of histone deacetylase in response to replication fork arrest and observed that in the presence of HU, the survival of cds1Δ cells was improved when the cells were simultaneously treated with histone deacetylase inhibitors. Furthermore, a mutation in the histone deacetylase gene, clr6, also suppresses the growth defect of cds1Δ cells in response to HU indicating a suppressive role of clr6-1 mutation in cds1 deletion background upon HU treatment. Interestingly, in response to HU, phosphorylation of Chk1 kinase and the number of Rad52YFP foci was reduced in cds1Δ clr6-1 double mutant as compared to cds1Δ single mutant indicating a decrease in the level of DNA damage in response to HU. Accordingly, the single-cell gel electrophoresis assay revealed a drastic reduction in the tail length of cds1Δ clr6-1 double mutant as compared to cds1Δ cells in the presence of HU suggesting the suppression of chromosomal defects in the double mutant. Taken together, we proposed that there could be transient suppression of fork collapse in cds1Δ clr6-1 double mutant upon HU treatment due to the delay in mitotic progression that leads to the facilitation of cell growth.

Objective Cisplatin, the most common chemotherapeutic drug for the treatment of advanced stage cervical cancers has limitations in terms of drugs resistance observed in patients partly due to functional DNA damage repair (DDR) processes in the cell. Mediator of DNA damage checkpoint 1 (MDC1) is an important protein in the Ataxia telangiectasia mutated (ATM) mediated double stranded DNA break (DSB) repair pathway. In this regard, we investigated the effect of MDC1 change in expression on the cisplatin sensitivity in cervical cancer cells. Results Through modulation of MDC1 expression in the cervical cancer cell lines; Hela, SiHa and Caski, we found that all the three cell lines silenced for MDC1 exhibited higher sensitivity to cisplatin treatment with inefficiency in accumulation of p γH2AX, Ser 139 foci and increased accumulation of pChk2 Thr 68 at the damaged chromatin followed by enhanced apoptosis. Further, we observed the increased p53 Ser 15 phosphorylation in the MDC1 depleted cells. Our studies suggest that MDC1 expression could be a key determinant in cervical cancer prognosis and its depletion in combination with cisplatin has the potential to be explored for the sensitisation of chemo-resistant cervical cancer cells.

Recently, RNAs interacting with proteins have been implicated in playing an important role in the occurrence and progression of oesophageal squamous cell carcinoma (ESCC). In this study, we found that NELFA mRNA interacts with Rad17 through a novel noncoding mode in the nucleus and that the aberrant expression of USF2 contributed to the upregulation of Rad17 and NELFA. Subsequent experiments demonstrated that the deletion of NELFA mRNA significantly decreased ESCC proliferation and colony formation in vitro. Moreover, NELFA mRNA knockdown inhibited DNA damage repair and promoted apoptosis. Mechanistic studies indicated that NELFA mRNA regulated the interaction between Rad17 and RFC2‐5, which had a major impact on the phosphorylation of CHK1, CHK2 and BRCA1. NELFA mRNA expression was consistently elevated in ESCC patients and closely related to decreased overall survival. Taken together, our results confirmed the critical role of the noncoding function of NELFA mRNA in ESCC tumorigenesis and indicated that NELFA mRNA can be regarded as a therapeutic target and an independent prognostic indicator in ESCC patients., NELFA mRNA interacts with Rad17 through a novel noncoding mode. NELFA mRNA knockdown significantly decreased oesophageal squamous cell carcinoma (ESCC) proliferation and colony formation. Moreover, NELFA mRNA knockdown inhibited DNA damage repair and promoted apoptosis. Mechanistic studies indicated that NELFA mRNA regulated the interaction between Rad17 and RFC2‐5. NELFA mRNA expression was consistently elevated in ESCC patients and closely related to decreased overall survival.

Background: Helicobacter pylori increases reactive oxygen species (ROS) and induces oxidative DNA damage and apoptosis in gastric epithelial cells. DNA damage activates DNA damage response (DDR) which includes ataxia-telangiectasia-mutated (ATM) activation. ATM increases alternative reading frame (ARF) but decreases mouse double minute 2 (Mdm2). Because p53 interacts with Mdm2, H. pylori–induced loss of Mdm2 stabilizes p53 and induces apoptosis. Previous study showed that Korean Red Ginseng extract (KRG) reduces ROS and prevents cell death in H. pylori–infected gastric epithelial cells. Methods: We determined whether KRG inhibits apoptosis by suppressing DDRs and apoptotic indices in H. pylori–infected gastric epithelial AGS cells. The infected cells were treated with or without KRG or an ATM kinase inhibitor KU-55933. ROS levels, apoptotic indices (cell death, DNA fragmentation, Bax/Bcl-2 ratio, caspase-3 activity) and DDRs (activation and levels of ATM, checkpoint kinase 2, Mdm2, ARF, and p53) were determined. Results: H. pylori induced apoptosis by increasing apoptotic indices and ROS levels. H. pylori activated DDRs (increased p-ATM, p-checkpoint kinase 2, ARF, p-p53, and p53, but decreased Mdm2) in gastric epithelial cells. KRG reduced ROS and inhibited increase in apoptotic indices and DDRs in H. pylori–infected gastric epithelial cells. KU-55933 suppressed DDRs and apoptosis in H. pylori–infected gastric epithelial cells, similar to KRG. Conclusion: KRG suppressed ATM-mediated DDRs and apoptosis by reducing ROS in H. pylori–infected gastric epithelial cells. Supplementation with KRG may prevent the oxidative stress-mediated gastric impairment associated with H. pylori infection. Keywords: Apoptosis, Gastric epithelial cells, Helicobacter pylori, Panax ginseng

Female reproductive success critically depends on the size and quality of a finite ovarian reserve. Paradoxically, mammals eliminate up to 80% of the initial oocyte pool through the enigmatic process of fetal oocyte attrition (FOA). Here, we interrogate the striking correlation of FOA with retrotransposon LINE-1 (L1) expression in mice to understand how L1 activity influences FOA and its biological relevance. We report that L1 activity triggers FOA through DNA damage-driven apoptosis and the complement system of immunity. We demonstrate this by combined inhibition of L1 reverse transcriptase activity and the Chk2-dependent DNA damage checkpoint to prevent FOA. Remarkably, reverse transcriptase inhibitor AZT-treated Chk2 mutant oocytes that evade FOA initially accumulate, but subsequently resolve, L1-instigated genotoxic threats independent of piRNAs and differentiate, resulting in an increased functional ovarian reserve. We conclude that FOA serves as quality control for oocyte genome integrity, and is not obligatory for oogenesis nor fertility., Mammals lose up to 80% of their finite oocyte supply during fetal development. Here the authors interrogate mechanisms of fetal oocyte attrition in mice, driven by the simultaneous upregulation of LINE-1 retrotransposon activity and inhibit these mechanisms to increase the functional ovarian reserve.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

We aimed to investigate the biological function of MELK and the therapeutic potential of OTSSP167 in human bladder cancer (BCa). First, we observed overexpression of MELK in BCa cell lines and tissues and found that it was associated with higher tumour stage and tumour grade, which was consistent with transcriptome analysis. High expression of MELK was significantly correlated with poor prognosis in BCa patients, and MELK was found to have a role in the cell cycle, the G1/S transition in mitosis, and DNA repair and replication. Furthermore, BCa cells presented significantly decreased proliferation capacity following silencing of MELK or treatment with OTSSP167 in vitro and in vivo. Functionally, reduction in MELK or treatment of cells with OTSSP167 could induce cell cycle arrest and could suppress migration. In addition, these treatments could activate phosphorylation of ATM and CHK2, which would be accompanied by down‐regulated MDMX, cyclin D1, CDK2 and E2F1; however, p53 and p21 would be activated. Opposite results were observed when MELK expression was induced. Overall, MELK was found to be a novel oncogene in BCa that induces cell cycle arrest via the ATM/CHK2/p53 pathway. OTSSP167 displays potent anti‐tumour activities, which may provide a new molecule‐based strategy for BCa treatment.

STUDY QUESTION Can we identify novel variants associated with polycystic ovary syndrome (PCOS) by leveraging the unique population history of Northern Europe? SUMMARY ANSWER We identified three novel genome-wide significant associations with PCOS, with two putative independent causal variants in the checkpoint kinase 2 (CHEK2) gene and a third in myosin X (MYO10). WHAT IS KNOWN ALREADY PCOS is a common, complex disorder with unknown aetiology. While previous genome-wide association studies (GWAS) have mapped several loci associated with PCOS, the analysis of populations with unique population history and genetic makeup has the potential to uncover new low-frequency variants with larger effects. STUDY DESIGN, SIZE, DURATION A population-based case–control GWAS was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS We identified PCOS cases from national registers by ICD codes (ICD-10 E28.2, ICD-9 256.4, or ICD-8 256.90), and all remaining women were considered controls. We then conducted a three-stage case–control GWAS: in the discovery phase, we had a total of 797 cases and 140 558 controls from the FinnGen study. For validation, we used an independent dataset from the Estonian Biobank, including 2812 cases and 89 230 controls. Finally, we performed a joint meta-analysis of 3609 cases and 229 788 controls from both cohorts. Additionally, we reran the association analyses including BMI as a covariate, with 2169 cases and 160 321 controls from both cohorts. MAIN RESULTS AND THE ROLE OF CHANCE Two out of the three novel genome-wide significant variants associating with PCOS, rs145598156 (P = 3.6×10−8, odds ratio (OR) = 3.01 [2.02–4.50] minor allele frequency (MAF) = 0.005) and rs182075939 (P = 1.9×10−16, OR = 1.69 [1.49–1.91], MAF = 0.04), were found to be enriched in the Finnish and Estonian populations and are tightly linked to a deletion c.1100delC (r2 = 0.95) and a missense I157T (r2 = 0.83) in CHEK2. The third novel association is a common variant near MYO10 (rs9312937, P = 1.7 × 10−8, OR = 1.16 [1.10–1.23], MAF = 0.44). We also replicated four previous reported associations near the genes Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4), DENN Domain Containing 1A (DENND1A), FSH Subunit Beta (FSHB) and Zinc Finger And BTB Domain Containing 16 (ZBTB16). When adding BMI as a covariate only one of the novel variants remained genome-wide significant in the meta-analysis (the EstBB lead signal in CHEK2 rs182075939, P = 1.9×10−16, OR = 1.74 [1.5–2.01]) possibly owing to reduced sample size. LARGE SCALE DATA The age- and BMI-adjusted GWAS meta-analysis summary statistics are available for download from the GWAS Catalog with accession numbers GCST90044902 and GCST90044903. LIMITATIONS, REASONS FOR CAUTION The main limitation was the low prevalence of PCOS in registers; however, the ones with the diagnosis most likely represent the most severe cases. Also, BMI data were not available for all (63% for FinnGen, 76% for EstBB), and the biobank setting limited the accessibility of PCOS phenotypes and laboratory values. WIDER IMPLICATIONS OF THE FINDINGS This study encourages the use of isolated populations to perform genetic association studies for the identification of rare variants contributing to the genetic landscape of complex diseases such as PCOS. STUDY FUNDING/COMPETING INTEREST(S) This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the MATER Marie Skłodowska-Curie grant agreement No. 813707 (N.P.-G., T.L., T.P.), the Estonian Research Council grant (PRG687, T.L.), the Academy of Finland grants 315921 (T.P.), 321763 (T.P.), 297338 (J.K.), 307247 (J.K.), 344695 (H.L.), Novo Nordisk Foundation grant NNF17OC0026062 (J.K.), the Sigrid Juselius Foundation project grants (T.L., J.K., T.P.), Finska Läkaresällskapet (H.L.) and Jane and Aatos Erkko Foundation (H.L.). The funders had no role in study design, data collection and analysis, publishing or preparation of the manuscript. The authors declare no conflicts of interest.

Aiming to identify rare high-penetrance mutations in new genes for the underlying predisposition in familial colorectal cancer (CRC), we performed whole-exome sequencing in 24 familial CRCs. Mutations in genes that regulate DNA repair (RMI1, PALB2, FANCI) were identified that were related to the Fanconi anemia DNA repair pathway. In one pedigree, we found a nonsense mutation in CHEK2. CHEK2 played an essential role in cell cycle and DNA damage repair. Somatic mutation analysis in CHEK2 variant carriers showed mutations in TP53, APC, and FBXW7. Loss of heterozygosity was found in carcinoma of CHEK2 variant carrier, and IHC showed loss of Chk2 expression in cancer tissue. We identified a second variant in CHEK2 in 126 sporadic CRCs. A KO cellular model for CHEK2 (CHEK2KO) was generated by CRISPR/Cas9. Functional experiments demonstrated that CHEK2KO cells showed defective cell cycle arrest and apoptosis, as well as reduced p53 phosphorylation, upon DNA damage. We associated germline mutations in genes that regulate the DNA repair pathway with the development of CRC. We identified CHEK2 as a regulator of DNA damage response and perhaps as a gene involved in CRC germline predisposition. These findings link CRC predisposition to the DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.

Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease. Hundreds of genetic loci associated with age at menopause, combined with experimental evidence in mice, highlight mechanisms of reproductive ageing across the lifespan.

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/., Author summary Male flies need to boost gene expression from their single X chromosome to equal that of females, which have two X chromosomes. In this process, called dosage compensation, the dosage compensation complex binds to genomic chromatin entry sites and upregulates gene expression nearby. This process was thought to be restricted to somatic cells. Using single-cell RNA-seq data, we found that certain germ cell types in the Drosophila testis show X chromosome expression similar to that of the autosomes, implying dosage compensation activity. In these cell types, we found evidence that genes near a chromatin entry site are more highly expressed than genes farther away, which is additional evidence of dosage compensation. In cell types without evidence of dosage compensation, we saw no evidence of chromatin entry site activity. Interestingly, we found little evidence of expression of most genes from the dosage compensation complex using both RNA-FISH and scRNA-seq. This suggests that our observed pre-meiotic dosage compensation is likely to be mediated by a noncanonical mechanism. These findings add new insight into our understanding of sex chromosomes.

Ionizing radiation (IR) induces DNA double-strand breaks that activate the DNA damage response (DDR), which leads to cell cycle arrest, senescence, or apoptotic cell death. Understanding the DDR of stem cells is critical to tissue homeostasis and the survival of the organism. Drosophila hematopoiesis serves as a model system for sensing stress and environmental changes; however, their response to DNA damage remains largely unexplored. The Drosophila lymph gland is the larval hematopoietic organ, where stem-like progenitors proliferate and differentiate into mature blood cells called hemocytes. We found that apoptotic cell death was induced in progenitors and hemocytes after 40 Gy irradiation, with progenitors showing more resistance to IR-induced cell death compared to hemocytes at a lower dose. Furthermore, we found that Drosophila ATM (tefu), Chk2 (lok), p53, and reaper were necessary for IR-induced cell death in the progenitors. Notably, IR-induced cell death in mature hemocytes required tefu, Drosophila JNK (bsk), and reaper, but not lok or p53. In summary, we found that DNA damage induces apoptotic cell death in the late third instar larval lymph gland and identified lok/p53-dependent and -independent cell death pathways in progenitors and mature hemocytes, respectively., Summary: Irradiation-induced cell death in larval lymph gland requires Drosophila ATM, Chk2, p53, and reaper in hematopoietic progenitors, while it involves Drosophila ATM, JNK, and reaper in differentiated blood cells.

Adenovirus (Ad) type 5 (Ad5) early region 4 (E4) proteins inhibit the DNA damage response (DDR) including activation of the DDR kinase ATM and its substrates, which can induce G2/M cell cycle arrest. Infection with Ad5 or the E4 deletion mutant H5dl1007 (1007) resulted in the accumulation of post G1 cells with > 2 N cellular DNA content. A greater fraction of cells with 4 N DNA content was observed in 1007 infections compared to Ad5; this population was dependent on activation of ATM. G2/M checkpoint kinases, phosphorylated Chk2 (pChk2), and phosphorylated Cdk1 (pCdk1) were upregulated in 1007 infections, and 1007 showed reduced levels of the mitosis marker phosphorylated (Ser10) histone 3 compared to Ad5. Our results show that E4 mutant activation of ATM induces G2/M arrest via activation of checkpoint kinases, thereby contributing to viral-mediated regulation of the cell cycle.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype., Author summary Cancer is a genetic disease caused by mutations that lead to uncontrolled cell proliferation and other tumour properties. Defects in DNA repair or replication can lead to cancer development by increasing the likelihood that cancer-causing mutations will happen. Here we look at a pathogenic variant of a polymerase involved in genome replication (DNA polymerase ε POLE-P286R). This variant is associated with highly mutated cancer genomes. By introducing this mutation into the polymerase ε gene of a model organism, fission yeast, we show that it causes a large increase in single base substitutions, scattered throughout the genome. The sequence context of mutations is similar in fission yeast and humans, suggesting that the yeast model is useful for understanding how POLE-P286R causes such a high mutation rate. Yeast POLE-P286R cells show slow chromosome replication, suggesting that the polymerase has difficulty in copying certain chromosomal regions. Yeast POLE-P286R cells become inviable when the concentration of dNTP building blocks for DNA synthesis is increased, probably because the mutation rate is pushed to an intolerable level. Interestingly, we find that specialised polymerases that are tolerant of DNA damage contribute to the high mutation rate caused by POLE-P286R. These findings have implications for the therapy of POLE-P286R tumours.

Proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in hepatocellular carcinoma; however, its underlying mechanism and role in hepatitis B virus (HBV) replication are largely unknown. Here, we found that HBV X protein (HBx) natural variants containing Ser-101 instead of Pro-101 increase reactive oxygen species levels in the mitochondria and activate the ataxia telangiectasia mutated/checkpoint kinase 2 pathway in the nucleus, resulting in the phosphorylation of p53 at Ser-15 and Ser-20 and the subsequent upregulation of its protein levels. Therefore, HBx variants containing Ser-101 induced p53-dependent activation of PA28γ expression in human hepatoma cells. The elevated PA28γ levels upregulated HBx levels through the inhibition of seven in absentia homologue 1-dependent proteasomal degradation. The self-amplifying ability of HBx variants containing Ser-101 via a positive feedback loop involving p53 and PA28γ was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, which also provided evidence for the stimulation of HBV replication by these HBx variants. In conclusion, the ability of HBx to upregulate PA28γ levels via p53 activation, in a Ser-101-dependent pathway, is critical for the stimulation of HBV replication.

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are important multifunctional proteins of the kinase family. Their main function is to regulate DNA replication and DNA damage response. If a cell is exposed to exogenous damage to its DNA, CHK1/CHK2 stops the cell cycle to give time to the cellular mechanisms to repair DNA breakage and apoptosis too, if the damage is not repairable to activate programmed cell death. CHK1/CHK2 plays a crucial role in the repair of recombination-mediated double-stranded DNA breaks. The other important functions performed by these proteins are the beginning of DNA replication, the stabilization of replication forks, the resolution of replication stress and the coordination of mitosis, even in the absence of exogenous DNA damage. Prexasertib (LY2606368) is a small ATP-competitive selective inhibitor of CHK1 and CHK2. In preclinical studies, prexasertib in monotherapy has shown to induce DNA damage and tumor cells apoptosis. The preclinical data and early clinical studies advocate the use of prexasertib in solid tumors both in monotherapy and in combination with other drugs (antimetabolites, PARP inhibitors and platinum-based chemotherapy). The safety and the efficacy of combination therapies with prexasertib need to be better evaluated in ongoing clinical trials.

Checkpoint kinase 2 (CHK-2) is a key component of the DNA damage response (DDR). CHK-2 is activated by the PIP3-kinase-like kinases (PI3KKs) ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR), and in metazoan also by DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These DNA damage-dependent activation pathways are conserved and additional activation pathways of CHK-2 are not known. Here we show that PERIOD-4 (PRD-4), the CHK-2 ortholog of Neurospora crassa, is part of a signaling pathway that is activated when protein translation is compromised. Translation stress induces phosphorylation of PRD-4 by a PI3KK distinct from ATM and ATR. Our data indicate that the activating PI3KK is mechanistic target of rapamycin (mTOR). We provide evidence that translation stress is sensed by unbalancing the expression levels of an unstable protein phosphatase that antagonizes phosphorylation of PRD-4 by mTOR complex 1 (TORC1). Hence, Neurospora mTOR and PRD-4 appear to coordinate metabolic state and cell cycle progression.

The 9-1-1 complex is a circular heterotrimeric complex composed of Rad9-Hus1-Rad1. In response to DNA damage, the 9-1-1 complex will be loaded onto the DNA damage site by clamp loader Rad24-RFC to activate the cell cycle checkpoint. The C-terminal of Ddc1/Rad9 is critical for checkpoint activation. However, there is little structural information about the intact 9-1-1 complex and the interaction with Rad24-RFC. Here, we determined the structure of the intact 9-1-1 complex in S. cerevisiae by cryo-Electron Microscopy (cryo-EM) and identified the Ddc1 C-tail module for the first time. We found that the C-terminal of Ddc1 has structural flexibility and it plays a critical role for Mec1/Ddc2 activation in G1/G2 phase. At the same time, we got a glimpse of the structure of Rad24-RFC and captured the interaction between the 9-1-1 complex and Rad24-RFC. The structural information greatly helped us to understand the process of clamp-loading.

Genetic variations in CHEK2 (checkpoint kinase 2) gene have been associated with hereditary predisposition to various cancers including breast and ovarian cancer. CHEK2 tumor suppressor gene encodes for a checkpoint kinase that responds to breaks in DNA, regulates DNA repair and cellular proliferation. We report a BRCA negative family with multiple affected women having breast cancer, with a novel, missense, likely pathogenic variant in the CHEK2 gene (c.1376T>G; p.Ile459Ser) that segregated with subjects with breast cancer. This case provides insight into the role of the CHEK2 gene in causing breast cancer susceptibility in families and supports the use of multigene panel testing in cases with hereditary predisposition to breast cancer.

The CHEK2 gene and its encoded protein Chk2 have a well-known role in cancers, especially those related to breast cancer mediated through the BRCA1 gene. Additionally Chk2 has a crucial role in DNA repair, apoptosis and the cell cycle, which is why classification of variants of uncertain significance (VUS) is an area highly sought for a better elucidation of the "genomic effect" that results. Because it can often take years before enough clinical data is accumulated, and the costly and expensive functional analysis for individual variants presents a significant hurdle, it is important to identify other tools to help aid in clarifying the impact of specific variants on a protein's function and eventually the patient's health outcome. Here we describe a newly identified CHEK2 variant and analyze with an integrated approach combining genomics (whole exome analysis), clinical study, radiographic imaging, and protein informatics to identify and predict the functional impact of the VUS on the protein's behavior and predicted impact on the related pathways. The observed and analyzed defects in the protein were consistent with the expected clinical effect. Here, we support the use of personalized protein modeling and informatics and further our goal of developing a large-scale protein deposition archive for all protein-level VUS.

Objective: Period1 (PER1), a core circadian gene, not only modulates circadian rhythm but may also play an important role in other biological processes, including pathways involved in the proliferation and apoptosis of tumor cells. In this study, we investigated the mechanism by which the downregulated expression of PER1 promotes the apoptosis of wild-type P53 human glioma U343 cells exposed to X-rays. Methods: U343 cells were exposed to 6 mV 10 Gy X-ray irradiation after infection with an shRNA lentivirus to reduce the expression of PER1 and were analyzed by SCGE analysis, flow cytometry, qRT-PCR, and western blotting. Result: SCGE analysis revealed that compared with the controls, U343 cells expressing low levels of PER1 showed minor DNA damage when exposed to X-ray irradiation (P

The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known. Here, we describe the separation-of-function sae2-ms mutant that, similar to SAE2 deletion, upregulates MRX and Tel1 signaling activities at DSBs by reducing Mre11 endonuclease activity. However, unlike SAE2 deletion, Sae2-ms causes neither DNA damage sensitivity nor enhanced Rad53 activation, indicating that DNA damage resistance depends mainly on Sae2-mediated Rad53 inhibition. The lack of Sae2, but not the presence of Sae2-ms, impairs long-range resection and increases both Rad9 accumulation at DSBs and Rad53–Rad9 interaction independently of Mre11 nuclease activity. Altogether, these data lead to a model whereby Sae2 plays distinct functions in limiting MRX-Tel1 and Rad9 abundance at DSBs, with the control on Rad9 association playing the major role in supporting DNA damage resistance and in regulating long-range resection and checkpoint activation.

Background/Aims: Excessive fluoride intake can induce cytotoxicity, DNA damage and cell-cycle changes in many tissues and organs, including the kidney. However, the underlying molecular mechanisms of fluoride-induced renal cell-cycle changes are not well understood at present. In this study, we used a mouse model to investigate how sodium fluoride (NaF) induces cell-cycle changes in renal cells. Methods: Two hundred forty ICR mice were randomly assigned to four equal groups for intragastric administration of NaF (0, 12, 24 and 48 mg/kg body weight/day) for 42 days. Kidneys were taken to measure changes of the cell-cycle at 21 and 42 days of the experiment, using flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot methods. Results: NaF, at more than 12 mg/kg body weight, induced G2/M phase cell-cycle arrest in the renal cells, which was supported by the finding of significantly increased percentages of renal cells in the G2/M phase. We found also that G2/M phase cell-cycle arrest was accompanied by up-regulation of p-ATM, p-Chk2, p-p53, p-Cdc25C, p-CDK1, p21, and Gadd45a protein expression levels; up-regulation of ATM, Chk2, p53, p21, and Gadd45a mRNA expression levels; down-regulation of CyclinB1, mdm2, PCNA protein expression levels; and down-regulation of CyclinB1, CDK1, Cdc25C, mdm2, and PCNA mRNA expression levels. Conclusion: In this mouse model, NaF, at more than 12 mg/ kg, induced G2/M phase cell-cycle arrest by activating the ATM-Chk2-p53/Cdc25C signaling pathway, which inhibits the proliferation of renal cells and development of the kidney. Activation of the ATM-Chk2-p53/Cdc25C signaling pathway is the mechanism of NaF-induced renal G2/M phase cell-cycle arrest in this model.

The highly conserved DNA damage response (DDR) pathway monitors the genomic integrity of the cell and protects against genotoxic stresses. The apical kinases, Mec1 and Tel1 (ATR and ATM in human, respectively), initiate the DNA damage signaling cascade through the effector kinases, Rad53 and Chk1, to regulate a variety of cellular processes including cell cycle progression, DNA damage repair, chromatin remodeling, and transcription. The DDR also regulates other cellular pathways, but direct substrates and mechanisms are still lacking. Using a mass spectrometry-based phosphoproteomic screen in Saccharomyces cerevisiae, we identified novel targets of Rad53, many of which are proteins that are involved in RNA metabolism. Of the 33 novel substrates identified, we verified that 12 are directly phosphorylated by Rad53 in vitro: Xrn1, Gcd11, Rps7b, Ded1, Cho2, Pus1, Hst1, Srv2, Set3, Snu23, Alb1, and Scp160. We further characterized Xrn1, a highly conserved 5′ exoribonuclease that functions in RNA degradation and the most enriched in our phosphoproteomics screen. Phosphorylation of Xrn1 by Rad53 does not appear to affect Xrn1’s intrinsic nuclease activity in vitro, but may affect its activity or specificity in vivo.

The 24-h circadian rhythm handles a wide variety of physiological needs. Clock genes, in coordination with other tissue-specific factors regulate various processes and often turns responsible for the pathological conditions when altered. Cancer is one such disease where the clock genes have been shown to contribute at multiple levels modulating key hallmarks of cancer. Most importantly, adding to this complication, noncoding RNAs (ncRNAs) have emerged as one of the major post-transcriptional regulators of gene expression and many recent studies have indicated about involvement of microRNAs or long noncoding RNAs in the process. In this review, we have described how do circadian pathway genes participated in oncogenesis and also updated the latest status of ncRNA involvement. We also try to address the existing gaps to have a more comprehensive understanding of the phenomenon in future.Abbreviations: HIFs: hypoxia-inducible factors; VEGF: Vascular endothelial growth factor; Mdm2: Mouse double minute 2 homolog; ATM: Ataxia telangiectasia mutated; Chk2: Checkpoint kinase 2; Bcl-Xl: B-cell lymphoma-extra-large; Bcl-2: B-cell lymphoma 2; DGCR8: DiGeorge syndrome chromosomal region 8; PPAR-γ: Peroxisome proliferator-activated receptor gamma.

Germline mutations of CHEK2 have been reported in various types of disease including breast cancer, ovarian cancer, colorectal cancer and prostate cancer. We generated two iPSC lines ZNHi001-A and ZNHi001-B from a prostate cancer patient carrying germline mutation in CHEK2 (c.667C＞T, also p.R223C) which may increase the risk of prostate cancer. Pluripotency and multi-lineage differentiation capacity of the two iPSC lines were confirmed by gene expression and teratoma assay. The generated iPSC lines carrying specific CHEK2 mutation might be a useful resource to study the pathogenic mechanism and develop potential therapeutic strategy of prostate cancer.

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

BACKGROUND: The G1 checkpoint is a critical regulator of genomic stability in untransformed cells, preventing cell cycle progression after DNA damage. DNA double-strand breaks (DSBs) recruit and activate ATM, a kinase which in turn activates the CHK2 kinase to establish G1 arrest. While the onset of G1 arrest is well understood, the specific role that ATM and CHK2 play in regulating G1 checkpoint maintenance remains poorly characterized. RESULTS: Here we examine the impact of ATM and CHK2 activities on G1 checkpoint maintenance in untransformed cells after DNA damage caused by DSBs. We show that ATM becomes dispensable for G1 checkpoint maintenance as early as 1h after DSB induction. In contrast, CHK2 kinase activity is necessary to maintain the G1 arrest, independently of ATM, ATR, and DNA-PKcs, implying that the G1 arrest is maintained in a lesion-independent manner. Sustained CHK2 activity is achieved through auto-activation and its acute inhibition enables cells to abrogate the G1-checkpoint and enter into S-phase. Accordingly, we show that CHK2 activity is lost in cells that recover from the G1 arrest, pointing to the involvement of a phosphatase with fast turnover. CONCLUSION: Our data indicate that G1 checkpoint maintenance relies on CHK2 and that its negative regulation is crucial for G1 checkpoint recovery after DSB induction. This research was funded by grants from MCIU/AEI/FEDER, UE (SAF2015-67562-R and RTI2018-097497-B-100) and Basque Government, Department of Education (IT1257-19) to A.M.Z., and Cancer Genomics Center Gravity Program (CGC.nl), Oncode Institute, Dutch Cancer Society (NKI 2014-6787) grants to R.H.M. I.G.-S. was supported with a postdoctoral fellowship from the Basque Country Government (Spain). J.V.-R. was supported by a postdoctoral fellowship from the University of the Basque Country (UPV/EHU).