Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

A subset of CB002 xanthine analogs bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53.

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes, including metastasis and therapy resistance. CB002 and structural-analogs restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2 checkpoint. Novel CB002-analogs induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogs target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression, and downregulation of essential proteins in DNA-synthesis and DNA-repair. CB002-analog #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs shows the activation of p53 pathway signaling in tumors with mutated p53, and targets an S-phase checkpoint.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.

Sulforaphane induces S-phase arrest and apoptosis via p53-dependent manner in gastric cancer cells.

Sulforaphane (SFN) extracted from broccoli sprout has previously been investigated for its potential properties in cancers, however, the underlying mechanisms of the anticancer activity of SFN remain not fully understood. In the present study, we investigate the effects of SFN on cell proliferation, cell cycle, cell apoptosis, and also the expression of several cell cycle and apoptosis-related genes by MTT assay, flow cytometry and western blot analysis in gastric cancer (GC) cells. The results showed that SFN could impair the colony-forming ability in BGC-823 and MGC-803 cell lines compared with the control. In addition, SFN significantly suppressed cell proliferation by arresting the cell cycle at the S phase and enhancing cell apoptosis in GC cells in a dose-dependent manner. Western blot results showed that SFN treatment significantly increased the expression levels of p53, p21 and decreased CDK2 expression, which directly regulated the S phase transition. The Bax and cleaved-caspase-3 genes involved in apoptosis executive functions were significantly increased in a dose-dependent manner in BGC-823 and MGC-803 cells. These results suggested that SFN-induced S phase cell cycle arrest and apoptosis through p53-dependent manner in GC cells, which suggested that SFN has a potential therapeutic application in the treatment and prevention of GC.

Roquin1 inhibits the proliferation of breast cancer cells by inducing G1/S cell cycle arrest via selectively destabilizing the mRNAs of cell cycle-promoting genes.

BACKGROUND: Dysregulation of cell cycle progression is a common feature of human cancer cells; however, its mechanism remains unclear. This study aims to clarify the role and the underlying mechanisms of Roquin1 in cell cycle arrest in breast cancer. METHODS: Public cancer databases were analyzed to identify the expression pattern of Roquin1 in human breast cancers and its association with patient survival. Quantitative real-time PCR and Western blots were performed to detect the expression of Roquin1 in breast cancer samples and cell lines. Cell counting, MTT assays, flow cytometry, and in vivo analyses were conducted to investigate the effects of Roquin1 on cell proliferation, cell cycle progression and tumor progression. RNA sequencing was applied to identify the differentially expressed genes regulated by Roquin1. RNA immunoprecipitation assay, luciferase reporter assay, mRNA half-life detection, RNA affinity binding assay, and RIP-ChIP were used to explore the molecular mechanisms of Roquin1. RESULTS: We showed that Roquin1 expression in breast cancer tissues and cell lines was inhibited, and the reduction in Roquin1 expression was associated with poor overall survival and relapse-free survival of patients with breast cancer. Roquin1 overexpression inhibited cell proliferation and induced G1/S cell cycle arrest without causing significant apoptosis. In contrast, knockdown of Roquin1 promoted cell growth and cycle progression. Moreover, in vivo induction of Roquin1 by adenovirus significantly suppressed breast tumor growth and metastasis. Mechanistically, Roquin1 selectively destabilizes cell cycle-promoting genes, including Cyclin D1, Cyclin E1, cyclin dependent kinase 6 (CDK6) and minichromosome maintenance 2 (MCM2), by targeting the stem-loop structure in the 3' untranslated region (3'UTR) of mRNAs via its ROQ domain, leading to the downregulation of cell cycle-promoting mRNAs. CONCLUSIONS: Our findings demonstrated that Roquin1 is a novel breast tumor suppressor and could induce G1/S cell cycle arrest by selectively downregulating the expression of cell cycle-promoting genes, which might be a potential molecular target for breast cancer treatment.

Cyclin Y binds and activates CDK4 to promote the G1/S phase transition in hepatocellular carcinoma cells via Rb signaling.

Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.

Long non­coding RNA SNHG12 regulates cell proliferation, invasion and migration in endometrial cancer by targeting miR­4429.

Long non­coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)­4429 were assessed in EC cell lines by reverse transcription­quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV­NC, miR­4429 mimic and mimic­NC, were transfected into RL95­2 cells. Post­transfection, Cell Counting Kit­8, Transwell Matrigel and wound­healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR­4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual­luciferase reporter system. Identified as a target of miR­4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR­4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR­4429 markedly suppressed proliferation, migration and invasion of RL95­2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR­4429 mimic­transfected RL95­2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR­4429 in EC. Therefore, targeting the SNHG12/miR­4429 axis could serve as a potential future therapeutic target for treatment of EC.

Genetic landscape and ligand-dependent activation of sonic hedgehog-Gli1 signaling in chordomas: a novel therapeutic target.

Chordoma, a rare neoplasm derived from intraosseous notochordal remnants, is unresponsive to conventional chemotherapy and radiotherapy. Sonic Hedgehog (Shh) is a crucial fetal notochord-secreted morphogen that directs notochordal development. The aim of this study was to determine the functional roles and therapeutic potential of Shh-Gli1 signaling in chordomas. Tissue samples and clinical profiles were collected from 42 patients with chordoma. The chordoma cell lines U-CH1 and MUG-Chor1 were used for functional experiments. Shh-Gli1 signaling pathway genetic alterations were screened, and the functions of the identified novel variants were analyzed using in silico analyses, real-time quantitative PCR, and minigene assays. Ligand-dependent Shh-Gli1 signaling activation was assessed using single- and dual-label immunostaining, western blot analysis, and a Shh-responsive Gli-luciferase reporter assay. The small-molecule inhibitor vismodegib was used to target Shh-Gli1 signaling in vitro and in vivo. Overall, 44 genetic alterations were identified, including four novel variants (c.67_69dupCTG in SMO, c.-6_-4dupGGC and c.3306 + 83_3306 + 84insG in PTCH1, and c.183-67_183-66delinsA in SUFU). Shh, PTCH1, SMO, SUFU, and Gli1 were extensively expressed in chordomas, and higher Gli1 expression correlated with poorer prognosis. A luciferase reporter assay and dual-label immunostaining indicated the occurrence of juxtacrine ligand-dependent Shh-Gli1 signaling activation. Vismodegib significantly inhibited cell proliferation and induced apoptosis and G1/S cell cycle arrest. In vivo investigation demonstrated that vismodegib effectively inhibited chordoma xenograft growth. This current preclinical evidence elucidates the therapeutic potential of Shh-Gli1 signaling pathway targeting for chordoma treatment. Vismodegib may be a promising targeted agent, and further clinical trials are warranted.

miR-144-3p inhibits cell proliferation of colorectal cancer cells by targeting BCL6 via inhibition of Wnt/ß-catenin signaling.

BACKGROUND: It has been shown that miR-144-3p regulates cell proliferation, apoptosis, migration and invasion in various cancers. However, the function and expression of miR-144-3p in colorectal cancer (CRC) remained obscure. METHODS: Immunohistochemical (IHC) staining was performed to investigate the protein expression of BCL6 in CRC tissues. The effect of BCL6 and miR-144-3p on CRC cells was explored through methylthiazolyl tetrazolium (MTT) assay, colony formation and cell cycle assays. Luciferase reporter assays, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were carried out to determine that BCL6 is directly regulated by miR-144-3p. RESULTS: Our results showed that miR-144-3p is down-regulated in CRC and correlates with the tumor progression of CRC patients. miR-144-3p inhibits cell proliferation and delays G1/S phase transition of CRC cells. Moreover, we found that BCL6 is a new target of miR-144-3p. Furthermore, BCL6 is a mediator of miR-144-3p repression of cell proliferation and cell cycle arrest in CRC cells. miR-144-3p repression of Wnt/ß-catenin signaling is mediated by BCL6 in CRC cells. CONCLUSIONS: Overall, the effect of the miR-144-3p/BCL6 axis on regulating CRC carcinogenesis was demonstrated, and miR-144-3p was identified as a potential prognostic and therapeutic target in colorectal cancer.

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

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

BAF45D knockdown decreases cell viability, inhibits colony formation, induces cell apoptosis and S-phase arrest in human pancreatic cancer cells.

Pancreatic cancer, an extremely aggressive malignancy, is resistant to chemo- or radiotherapy. The rapid progression of pancreatic cancer without distinctive clinical sign makes early diagnosing and/or treating very difficult. BAF45D, a member of the d4 domain family, is involved in oncogenic processes. However, the role of BAF45D in pancreatic tumorigenesis is largely unclear. Our goal is to examine BAF45D protein expression after lentivirus-mediated Baf45d RNAi and explore the effects of BAF45D knockdown on cell proliferation, cell apoptosis, and cell cycle of human pancreatic cancer cells. Here our results showed that Baf45d RNAi downregulated BAF45D protein levels and decreased cell viability, increased cell apoptosis, and decreased colony formation in BxPC-3 cells. Moreover, BAF45D knockdown induced S-phase arrest in BxPC-3 cells. Our results here suggest that BAF45D may play a crucial role in tumorigenic properties of human pancreatic cancer cells.

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress.

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Safe and Efficacious Diphtheria Toxin-Based Treatment for Melanoma: Combination of a Light-On Gene-Expression System and Nanotechnology.

The controversy surrounding the use of diphtheria toxin (DT) as a therapeutic agent against tumor cells arises mainly from its unexpected harmfulness to healthy tissues. We encoded the cytotoxic fragment A of DT (DTA) as an objective gene in the Light-On gene-expression system to construct plasmids pGAVPO (pG) and pU5-DTA (pDTA). Meanwhile, a cRGD-modified ternary complex comprising plasmids, chitosan, and liposome (pG&pDTA@cRGD-CL) was prepared as a nanocarrier to ensure transfection efficiency. Benefiting from spatiotemporal control of this light-switchable transgene system and the superior tumor targeting of the carrier, toxins were designed to be expressed selectively in illuminated lesions. In vitro studies suggested that pG&pDTA@cRGD-CL exerted arrest of the S phase in B16F10 cells upon blue light irradiation and, ultimately, induced the apoptosis and necrosis of tumor cells. Such DTA-based treatment exerted enhanced antitumor activity in mice bearing B16F10 xenografts and displayed prolonged survival time with minimal side effects. Hence, we described novel DTA-based therapy combined with nanotechnology and the Light-On gene-expression system: such treatment could be a promising strategy against melanoma.

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

WNT5B exerts oncogenic effects and is negatively regulated by miR-5587-3p in lung adenocarcinoma progression.

WNT5B glycoprotein belongs to the Wnt protein family. Limited investigations revealed a possible role of WNT5B in malignancies, such as triple-negative breast cancer and oral squamous cell carcinoma. However, whether WNT5B contributes to the progression of lung adenocarcinoma (LAD) remains unclear. Here, we initially determine that WNT5B is highly expressed in LAD and is positively correlated with lymph node metastasis and TNM stage. Consistently, clinical analysis reveals WNT5B as an independent prognostic biomarker in LAD. Silencing WNT5B suppresses the proliferation of LAD both in vitro and in vivo by interfering G1/S cell-cycle progression and modulating amino acid metabolism, revealing its remarkable oncogenic role in LAD. Of note, we also identified miR-5587-3p as a negative upstream regulator of WNT5B in LAD, which may help develop therapies targeting LAD patients with high WNT5B expression. Taken together, our results revealed an oncogenic role of WNT5B in LAD, which could be a prognostic biomarker and promising therapeutic target for LAD patients.

TOMM20 as a potential therapeutic target of colorectal cancer.

Translocase of outer mitochondrial membrane 20 (TOMM20) plays an essential role as a receptor for proteins targeted to mitochondria. TOMM20 was shown to be overexpressed in various cancers. However, the oncological function and therapeutic potential for TOMM20 in cancer remains largely unexplored. The purpose of this study was to elucidate the underlying molecular mechanism of TOMM20's contribution to tumorigenesis and to explore the possibility of its therapeutic potential using colorectal cancer as a model. The results show that TOMM20 overexpression resulted in an increase in cell proliferation, migration, and invasion of colorectal cancer (CRC) cells, while siRNA-mediated inhibition of TOMM20 resulted in significant decreases in cell proliferation, migration, and invasion. TOMM20 expression directly impacted the mitochondrial function including ATP production and maintenance of membrane potential, which contributed to tumorigenic cellular activities including regulation of S phase cell cycle and apoptosis. TOMM20 was overexpressed in CRC compared to the normal tissues and increased expression of TOMM20 to be associated with malignant characteristics including a higher number of lymph nodes and perineural invasion in CRC. Notably, knockdown of TOMM20 in the xenograft mouse model resulted in a significant reduction of tumor growth. This is the first report demonstrating a relationship between TOMM20 and tumorigenesis in colorectal cancer and providing promising evidence for the potential for TOMM20 to serve as a new therapeutic target of colorectal cancer. [BMB Reports 2019; 52(12): 712-717].

A novel system of bacterial cell division arrest implicated in horizontal transmission of an integrative and conjugative element.

Integrative and conjugative elements (ICEs) are widespread mobile DNA elements in the prokaryotic world. ICEs are usually retained within the bacterial chromosome, but can be excised and transferred from a donor to a new recipient cell, even of another species. Horizontal transmission of ICEclc, a prevalent ICE in proteobacteria, only occurs from developed specialized transfer competent (tc) cells in the donor population. tc cells become entirely dedicated to the ICE transmission at the cost of cell proliferation. The cell growth impairment is mediated by two ICEclc located genes, parA and shi, but the mechanistic and dynamic details of this process are unknown. To better understand the function of ParA and Shi, we followed their intracellular behavior from fluorescent protein fusions, and studied host cell division at single-cell level. Superresolution imaging revealed that ParA-mCherry colocalized with the host nucleoid while Shi-GFP was enriched at the membrane during the growth impairment. Despite being enriched at different cellular locations, the two proteins showed in vivo interactions, and mutations in the Walker A motif of ParA dislocalized both ParA and Shi. In addition, ParA mutations in the ATPase motif abolished the growth arrest on the host cell. Time-lapse microscopy revealed that ParA and Shi initially delay cell division, suggesting an extension of the S phase of cells, but eventually completely inhibit cell elongation. The parA-shi locus is highly conserved in other ICEclc-related elements, and expressing ParA-Shi from ICEclc in other proteobacterial species caused similar growth arrest, suggesting that the system functions similarly across hosts. The results of our study provide mechanistic insight into the novel and unique system on ICEs and help to understand such epistatic interaction between ICE genes and host physiology that entails efficient horizontal gene transfer.

Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA.

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.