DNA Replication Licensing Factor Research Articles

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4–TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4–TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4–Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Identification of key genes and molecular pathways associated with claw regeneration in mud crab (Scylla paramamosain)

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration.

Glutamine Regulates Gene Expression Profiles to Increase the Proliferation of Porcine Intestinal Epithelial Cells and the Expansion of Intestinal Stem Cells.

The intestinal epithelium is known for its rapid self-renewal, and glutamine is crucial in providing carbon and nitrogen for biosynthesis. However, understanding how glutamine affects gene expression in the intestinal epithelium is limited, and identifying the essential genes and signals involved in regulating intestinal epithelial cell growth is particularly challenging. In this study, glutamine supplementation exhibited a robust acceleration of intestinal epithelial cell proliferation and stem cell expansion. RNA sequencing indicated diverse transcriptome changes between the control and glutamine supplementation groups, identifying 925 up-regulated and 1152 down-regulated genes. The up-regulated DEGs were enriched in the KEGG pathway of cell cycle and GO terms of DNA replication initiation, regulation of phosphatidylinositol 3-kinase activity, DNA replication, minichromosome maintenance protein (MCM) complex, and ATP binding, whereas the down-regulated DEGs were enriched in the KEGG pathway of p53 signaling pathway, TNF signaling pathway, and JAK-STAT signaling pathway and GO terms of inflammatory response and intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress. Furthermore, GSEA analysis revealed a significant up-regulation of the cell cycle, DNA replication initiation, ATP-dependent RNA helicase activity, and down-regulation of the TNF signaling pathway. The protein-protein association network of the intersecting genes highlighted the significance of DNA replication licensing factors (MCM3, MCM6, and MCM10) in promoting intestinal epithelial growth in response to glutamine. Based on these findings, we propose that glutamine may upregulate DNA replication licensing factors, leading to increased PI3K/Akt signaling and the suppression of TNF, JAK-STAT, and p53 pathways. Consequently, this mechanism results in the proliferation of porcine intestinal epithelial cells and the expansion of intestinal stem cells.

The Ndc80-Cdt1-Ska1 complex is a central processive kinetochore-microtubule coupling unit.

It is known that microtubule-binding proteins including the Ska1 complex and the DNA replication licensing factor, Cdt1, enable the kinetochore-localized Ndc80 complex to form robust kinetochore-microtubule attachments. However, it is not clear how the Ndc80 complex is stably coupled to dynamic spindle microtubule plus-ends. Here, we have developed a conditional auxin-inducible degron approach to reveal a function for Cdt1 in chromosome segregation and kinetochore-microtubule interactions that is separable from its role in DNA replication licensing. Further, we demonstrate that a direct interaction between Cdt1 and Ska1 is required for recruiting Cdt1 to kinetochores and spindle microtubules. Cdt1 phosphorylation by Cdk1 kinase is critical for Ska1 binding, kinetochore-microtubule attachments, and mitotic progression. Furthermore, we show that Cdt1 synergizes with Ndc80 and Ska1 for microtubule binding, including forming a diffusive, tripartite Ndc80-Cdt1-Ska1 complex that can processively track dynamic microtubule plus-ends in vitro. Taken together, our data identify the Ndc80-Cdt1-Ska1 complex as a central molecular unit that can promote processive bidirectional tip-tracking of microtubules by kinetochores.

Pharmacokinetics and drug-likeness of anti-cancer traditional Chinese medicine: molecular docking and molecular dynamics simulation study

MCM7 (Minichromosome Maintenance Complex Component 7) is a component of the DNA replication licensing factor, which controls DNA replication. The MCM7 protein is linked to tumor cell proliferation and has a function in the development of several human cancers. Several types of cancer may be treated by inhibiting the protein, as it is strongly produced throughout this process. Significantly, Traditional Chinese Medicine (TCM), which has a long history of clinical adjuvant use against cancer, is rapidly gaining traction as a valuable medical resource for the development of novel cancer therapies, including immunotherapy. Therefore, the goal of the research was to find small molecular therapeutic candidates against the MCM7 protein that may be used to treat human cancers. A computational-based virtual screening of 36,000 natural TCM libraries is carried out for this goal using a molecular docking and dynamic simulation technique. Thereby, ∼8 novel potent compounds i.e., ZINC85542762, ZINC95911541, ZINC85542617, ZINC85542646, ZINC85592446, ZINC85568676, ZINC85531303, and ZINC95914464 were successfully shortlisted, each having the capacity to penetrate the cell as potent inhibitors for MCM7 to curb this disorder. These selected compounds were found to have high binding affinities compared to the reference (AGS compound) i.e. < −11.0 kcal/mol. ADMET and pharmacological properties showed that none of these 8 compounds poses any toxic property (carcinogenicity) and have anti-metastatic, and anticancer activity. Additionally, MD simulations were run to assess the compounds’ stability and dynamic behavior with the MCM7 complex for about 100 ns. Finally, ZINC95914464, ZINC95911541, ZINC85568676, ZINC85592446, ZINC85531303, and ZINC85542646 are identified as highly stable within the complex throughout the 100 ns simulations. Moreover, the results of binding free energy suggested that the selected virtual hits significantly bind to the MCM7 which implied these compounds may act as a potential MCM7 inhibitor. However, in vitro testing protocols are required to further support these results. Further, assessment through various lab-based trial methods can assist with deciding the action of the compound that will give options in contrast to human cancer immunotherapy. Communicated by Ramaswamy H. Sarma

SPOP is essential for DNA replication licensing through maintaining translation of CDT1 and CDC6 in HaCaT cells

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.

Integrated transcriptome and proteome revealed that the declined expression of cell cycle-related genes associated with follicular atresia in geese

BackgroundGeese exhibit relatively low reproductive performance, and follicular atresia is an important factor that restricts the egg production of geese. Systematic analysis of the regulation of follicle atresia in geese through transcriptome and proteome levels could provide meaningful information on clarifying the mechanism of follicle atresia in poultry.ResultThe granulosa cell layer was loose, disintegrated and showed apoptosis in atretic follicles and remained intact in normal follicles. The hormone levels of FSH and LH were significantly decreased in the atresia follicles compared to the normal follicles (P < 0.05). A total of 954 differentially expressed genes (DEGs, 315 increased and 639 decreased) and 161 differentially expressed proteins (DEPs, 61 increased and 100 decreased) were obtained in atresia follicles compared to normal follicles, of which, 15 genes were differentially expressed in both transcriptome and proteome. The DEGs were mainly enriched in sodium transmembrane transport, plasma membrane, and transmembrane transporter activity based on the GO enrichment analysis and in the cell cycle pathway based on the KEGG enrichment analysis. The DEPs were mainly enriched in localization, lysosome, and phospholipid-binding based on the GO enrichment analysis. Candidate genes Smad2/3, Smad4, Annexin A1 (ANXA1), Stromelysin-1 (MMP3), Serine/threonine-protein kinase (CHK1), DNA replication licensing factor (MCM3), Cyclin-A2 (CCNA2), mitotic spindle assembly checkpoint protein (MAD2), Cyclin-dependent kinase 1 (CDK1), fibroblast growth factor 12 (FGF12), and G1/S-specific cyclin-D1 (CCND1) were possibly responsible for the regulation of atresia.ConclusionThe cell cycle is an important pathway for the regulation of follicular atresia. Sodium outflow and high expression of MMP3 and MMP9 could be responsible for structural destruction and apoptosis of follicular cells.

An autoantigen profile from Jurkat T-Lymphoblasts provides a molecular guide for investigating autoimmune sequelae of COVID-19

In order to understand autoimmune phenomena contributing to the pathophysiology of COVID-19 and post-COVID syndrome, we have been profiling autoantigens (autoAgs) from various cell types. Although cells share numerous autoAgs, each cell type gives rise to unique COVID-altered autoAg candidates, which may explain the wide range of symptoms experienced by patients with autoimmune sequelae of SARS-CoV-2 infection. Based on the unifying property of affinity between autoAgs and the glycosaminoglycan dermatan sulfate (DS), this paper reports 140 candidate autoAgs identified from proteome extracts of human Jurkat T-cells, of which at least 105 (75%) are known targets of autoantibodies. Comparison with currently available multi-omic COVID-19 data shows that 125 (89%) DS-affinity proteins are altered at protein and/or RNA levels in SARS-CoV-2-infected cells or patients, with at least 94 being known autoAgs in a wide spectrum of autoimmune diseases and cancer. Protein alterations by ubiquitination and phosphorylation during the viral infection are major contributors of autoAgs. The autoAg protein network is significantly associated with cellular response to stress, apoptosis, RNA metabolism, mRNA processing and translation, protein folding and processing, chromosome organization, cell cycle, and muscle contraction. The autoAgs include clusters of histones, CCT/TriC chaperonin, DNA replication licensing factors, proteasome and ribosome proteins, heat shock proteins, serine/arginine-rich splicing factors, 14-3-3 proteins, and cytoskeletal proteins. AutoAgs, such as LCP1 and NACA, that are altered in the T cells of COVID patients may provide insight into T-cell responses to viral infection and merit further study. The autoantigen-ome from this study contributes to a comprehensive molecular map for investigating acute, subacute, and chronic autoimmune disorders caused by SARS-CoV-2.

Genomic analyses provide insights into the evolution and salinity adaptation of halophyte Tamarix chinensis.

The woody halophyte Tamarix chinensis is a pioneer tree species in the coastal wetland ecosystem of northern China, exhibiting high resistance to salt stress. However, the genetic information underlying salt tolerance in T. chinensis remains to be seen. Here we present a genomic investigation of T. chinensis to elucidate the underlying mechanism of its high resistance to salinity. Using a combination of PacBio and high-throughput chromosome conformation capture data, a chromosome-level T. chinensis genome was assembled with a size of 1.32 Gb and scaffold N50 of 110.03 Mb. Genome evolution analyses revealed that T. chinensis significantly expanded families of HAT and LIMYB genes. Whole-genome and tandem duplications contributed to the expansion of genes associated with the salinity adaptation of T. chinensis. Transcriptome analyses were performed on root and shoot tissues during salt stress and recovery, and several hub genes responding to salt stress were identified. WRKY33/40, MPK3/4, and XBAT31 were critical in responding to salt stress during early exposure, while WRKY40, ZAT10, AHK4, IRX9, and CESA4/8 were involved in responding to salt stress during late stress and recovery. In addition, PER7/27/57/73 encoding class III peroxidase and MCM3/4/5/7 encoding DNA replication licensing factor maintained up/downregulation during salt stress and recovery stages. The results presented here reveal the genetic mechanisms underlying salt adaptation in T. chinensis, thus providing important genomic resources for evolutionary studies on tamarisk and plant salt tolerance genetic improvement.

Comparative multiomics analysis of cell physiological state after culture in a basket bioreactor

Bioreactors are one of the most important, basic pieces of equipment in the biopharmaceutical industry. Understanding the effects of mechanical damage and other factors on the physiological state of cells during cell matrix culture is the basis for continuously achieving greater efficiency and higher product quality. In this study, Vero cells were used as a model and apoptosis, senescence, transcriptomics, proteomics, and metabolomics were carried out for analysis at the cellular and molecular levels. The results showed that compared with cells cultured in the simulated natural state, the cells cultured in the basket bioreactor displayed no obvious senescence. Additionally, the proportion of early apoptotic cells increased, but the proportions of damaged, late apoptotic and dead cells did not change significantly. The transcription levels of aminoacyl-tRNA synthetase and cyclin D1 and the expression levels of DNA replication licensing factor, methenyltetrahydrofolate cyclohydrolase, arachidonic acid and other metabolites of cells cultured in the basket bioreactor were significantly increased. These results suggest that DNA replication, protein translation and the metabolic activities in cells cultured in basket bioreactors are more active, which is more conducive to cell amplification and target product production. In this study, the growth and physiological state of cells in a basket bioreactor were characterized at the molecular level for the first time. Additionally, a tool to evaluate the physiological state of cells in a bioreactor was established, which can be used to guide the development and optimization of cell matrix culture conditions in industrial production and improve the production efficiency of the target products.

Integrated physiological, metabolomic, and proteome analysis of Alpinia officinarum Hance essential oil inhibits the growth of Fusarium oxysporum of Panax notoginseng.

Fusarium oxysporum is the main pathogen of Panax notoginseng root rot, and chemical fungicides remain the primary measures to control the disease. Plant essential oil (EO) is a volatile plant secondary metabolic product that does not produce any residue to replace chemical pesticide. To comprehensively understand the antifungal mechanism of Alpinia officinarum Hance EO, the physiological indicators, proteome and metabolome were analyzed using F. oxysporum spores and hyphae treated with different EO concentrations. The cell membrane was damaged after both low and high concentrations of EO treatment, along with leakage of the cell contents. To resist the destruction of membrane structure, fungi can increase the function of steroid biosynthesis and expression of these catalytic enzymes, including squalene monooxygenase (SQLE), sterol 14alpha-demethylase (CYP51, CYP61A), delta14-sterol reductase (TM7SF2, ERG4), methylsterol monooxygenase (MESO1), and sterol 24-C-methyltransferase (SMT1). Furthermore, the tricarboxylic acid cycle (TCA) was influenced by inhibiting the expression of glutamate synthase (GLT1), 4-aminobutyrate aminotransferase (ABAT), and succinate-semialdehyde dehydrogenase (gabD); increasing malate and gamma-aminobutyric acid (GABA); and decreasing citrate content. The spore germination rate and mycelia growth were decreased because the expression of cohesin complex subunit SA-1/2 (IRR1) and cohesion complex subunit (YCS4, BRN1, YCG1) were inhibited. Particularly, under high EO concentrations, cyclin-dependent kinase (CDC28) and DNA replication licensing factor (MCM) were further inhibited to disrupt the cell cycle and meiosis, thus affecting cell division. The results of this study will enrich the understanding of the antifungal mechanism of EOs and provide an important basis to develop new plant-derived fungicides.

Unveiling Molecular Mechanisms of Nitric Oxide-Induced Low-Temperature Tolerance in Cucumber by Transcriptome Profiling.

Cucumber (Cucumis sativus L.) is one of the most popular cultivated vegetable crops but it is intrinsically sensitive to cold stress due to its thermophilic nature. To explore the molecular mechanism of plant response to low temperature (LT) and the mitigation effect of exogenous nitric oxide (NO) on LT stress in cucumber, transcriptome changes in cucumber leaves were compared. The results showed that LT stress regulated the transcript level of genes related to the cell cycle, photosynthesis, flavonoid accumulation, lignin synthesis, active gibberellin (GA), phenylalanine metabolism, phytohormone ethylene and salicylic acid (SA) signaling in cucumber seedlings. Exogenous NO improved the LT tolerance of cucumber as reflected by increased maximum photochemical efficiency (Fv/Fm) and decreased chilling damage index (CI), electrolyte leakage and malondialdehyde (MDA) content, and altered transcript levels of genes related to phenylalanine metabolism, lignin synthesis, plant hormone (SA and ethylene) signal transduction, and cell cycle. In addition, we found four differentially expressed transcription factors (MYB63, WRKY21, HD-ZIP, and b-ZIP) and their target genes such as the light-harvesting complex I chlorophyll a/b binding protein 1 gene (LHCA1), light-harvesting complex II chlorophyll a/b binding protein 1, 3, and 5 genes (LHCB1, LHCB3, and LHCB5), chalcone synthase gene (CSH), ethylene-insensitive protein 3 gene (EIN3), peroxidase, phenylalanine ammonia-lyase gene (PAL), DNA replication licensing factor gene (MCM5 and MCM6), gibberellin 3 beta-dioxygenase gene (GA3ox), and regulatory protein gene (NPRI), which are potentially associated with plant responses to NO and LT stress. Notably, HD-ZIP and b-ZIP specifically responded to exogenous NO under LT stress. Taken together, these results demonstrate that cucumber seedlings respond to LT stress and exogenous NO by modulating the transcription of some key transcription factors and their downstream genes, thereby regulating photosynthesis, lignin synthesis, plant hormone signal transduction, phenylalanine metabolism, cell cycle, and GA synthesis. Our study unveiled potential molecular mechanisms of plant response to LT stress and indicated the possibility of NO application in cucumber production under LT stress, particularly in winter and early spring.

First Report of Berkeleyomyces basicola Causing Black Root Rot on Lisianthus (Eustoma grandiflorum) in China.

Lisianthus (Eustoma grandiflorum (Raf.) Shinn.) is an important ornamental plant ranking in the top 10 cut flowers worldwide (Xiao et al., 2018). In 2020 and 2021, black root rot was found as a major disease limiting lisianthus production in Yunnan Province, China. Black root rot was first observed in early July 2020 on lisianthus grown in a commercial flower-production plantation, with nearly 60% plants infected. Symptoms appeared as coalescing necrotic lesions leading to black discoloration of the roots. Root damage induced by disease resulted in insufficient water and nutrient uptake by the plant, causing stunting and whole-plant wilting. The pathogen could not infect the intact endodermis, and vascular tissues below the discolored cortical tissue remained healthy. Symptomatic roots were surface sterilized using 1% NaClO for 1 min, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C for 7 days in the dark. The morphological characteristics were basically consistent: the colonies were white to gray in color, and the conidiophores were colorless to brown, solitary or clustered. Conidia were single-celled, colorless, rod-shaped, and obtuse at both ends. Chlamydospores were dark brown, clustered or solitary. The morphological characteristics of the pathogen were similar to those of Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong & M.J. Wingf. (Nakane et al. 2019). DNA was extracted from mycelia of representative isolate TB using the Plant Genomic DNA Kit (Tiangen, Beijing, China). The internal transcribed spacers (ITS), DNA replication licensing factor (MCM7), ribosomal large subunit (LSU), and 60S ribosomal protein RPL10 (60S) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), MCM7-for/MCM7-rev, LR0R/LR5, and 60S-506F/60S-908R, respectively (Nel et al. 2018). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolate (TB) and three published sequences of B. basicola were clustered into one clade with a 100% bootstrapping value. The accession numbers of B. basicola reference sequences are MF952423 (ITS), MF967079 (MCM7), MF948658 (LSU), and MF967072 (60S) of isolate CMW6714; MF952428 (ITS), MF967088 (MCM7), MF948661 (LSU), and MF967073 (60S) of isolate CMW25440; MF952429 (ITS), MF967102 (MCM7), MF948659 (LSU), and MF967075 (60S) of isolate CMW49352. The sequences of TB have been deposited in GenBank with accession numbers MZ351733 for ITS, MZ695817 for MCM7, MZ695816 for LSU, and MZ695815 for the 60S region. To verify the pathogenicity of the fungus, inoculations were performed on ten 2-month-old potted lisianthus plants by dipping the roots into a conidial suspension (105 spores/ml) for 2 h. Ten plants were mock inoculated with distilled water as a control. Symptoms of black root rot were observed 30 days after inoculation, whereas the control roots remained healthy. The causal fungus has a host range of over 230 species and is a destructive pathogen of many crops and ornamental plants, including cotton (Gossypium barbadense L.), tobacco (Nicotiana tabacum L.) and mango (Mangifera indica L.) (Shukla et al. 2021; Toksoz and Rothrock 2009). This is the first report worldwide of B. basicola infecting lisianthus. This discovery is of great importance for Chinese flower growers because this fungus is well established in the observed area, and effective measures are needed to manage this disease.

Bioinformatics approaches identified dasatinib and bortezomib inhibit the activity of MCM7 protein as a potential treatment against human cancer

Minichromosome Maintenance Complex Component 7 (MCM7) is a key component of the DNA replication licensing factor and hexamer MCM (MCM2–7) complex that regulates the DNA replication process. The MCM7 protein is associated with tumor cell proliferation that plays an important role in different human cancer progression. As the protein is highly expressed during the cancer development process, therefore, inhibition of the protein can be utilized as a treatment option for different human cancer. However, the study aimed to identify potential small molecular drug candidates against the MCM7 protein that can utilize treatment options for human cancer. Initially, the compounds identified from protein-drugs network analysis have been retrieved from NetworkAnalyst v3.0 server and screened through molecular docking, MM-GBSA, DFT, pharmacokinetics, toxicity, and molecular dynamics (MD) simulation approach. Two compounds namely Dasatinib (CID_3062316) and Bortezomib (CID_387447) have been identified throughout the screening process, which have the highest negative binding affinity (Kcal/mol) and binding free energy (Kcal/mol). The pharmacokinetics and toxicity analysis identified drug-like properties and no toxicity properties of the compounds, where 500 ns MD simulation confirmed structural stability of the two compounds to the targeted proteins. Therefore, we can conclude that the compounds dasatinib and bortezomib can inhibit the activity of the MCM7 and can be developed as a treatment option against human cancer.

DNA Replication Licensing Factors: Novel Targets for Cancer Therapy via Inhibiting the Stemness of Cancer Cells.

The replication licensing factors strictly regulate the DNA replication origin licensing process to guarantee the stability of the genome. Numerous experimental studies have recently demonstrated that the replication licensing factors as oncogenes are essential for the occurrence and development of cancers. Drug resistance, being one of the main characteristics of cancer stem cells, can cause a high recurrence rate and a low survival rate in patients with different cancers. However, the function of the replication licensing factors in cancer stemness remains unclear. The following article highlights the most recent research on DNA replication origin licensing factors in cancer and their function in anti-cancer drug resistance. Moreover, this article proposes a new perspective that replication licensing factors as chemotherapy shield affect anti-cancer drug resistance by promoting the stemness of cancer cells.

Molecular determinants of phase separation for Drosophila DNA replication licensing factors.

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection

BackgroundThe Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation.ResultsIn this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells.ConclusionsOur findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

Clinicopathologic significance of DNA replication licensing factors in head and neck diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) harbors defects in the proliferation pathway. We performed multiparameter analysis of proteins expressed during different cell cycle phases and correlated them with clinical parameters of head and neck DLBCLs. Thirty-nine DLBCLs were staged and immunohistochemically stained with MCM2, Ki67, and geminin. The receiver operating characteristic curve and its area under the curve were calculated, and sensitivity vs specificity curve analysis was performed. The highest labeling index was in MCM2, followed by Ki67 and geminin (P < .001). All pairs showed significant differences (P < .001). The best cutoff points to differentiate limited from advanced disease were 68% and 45% for MCM2 and Ki67, respectively. There was no acceptable cutoff for geminin (area under the curve=0.667, P=.134). MCM2/Ki67 (P=.293) and geminin/Ki67 (P=.233) ratios did not differ between the stages. The median (interquartile range) of the geminin/Ki67 ratio was 0.57 (0.68), translating to a reduced G1. We suggest a role for cell cycle-related proteins in the biology and behavior of DLBCLs. MCM2 and Ki67 cutoffs can be a potential option to differentiate limited from advanced disease, where imaging and laboratory techniques are unavailable. The G1 decrease and the significantly higher MCM2 expression compared to Ki67 indicate replication disturbances, making factors involved in the G1 phase targets for treatment.

Lysophosphatidic Acid-Induced EGFR Transactivation Promotes Gastric Cancer Cell DNA Replication by Stabilizing Geminin in the S Phase.

Geminin, an inhibitor of the DNA replication licensing factor, chromatin licensing and DNA replication factor (Cdt) 1, is essential for the maintenance of genomic integrity. As a multifunctional protein, geminin is also involved in tumor progression, but the molecular details are largely unknown. Here, we found that lysophosphatidic acid (LPA)–induced upregulation of geminin was specific to gastric cancer cells. LPA acted via LPA receptor (LPAR) 3 and matrix metalloproteinases (MMPs) signaling to transactivate epidermal growth factor receptor (EGFR) (Y1173) and thereby stabilize geminin expression level during the S phase. LPA also induced the expression of deubiquitinating protein (DUB) 3, which prevented geminin degradation. These results reveal a novel mechanism underlying gastric cancer progression that involves the regulation of geminin stability by LPA-induced EGFR transactivation and provide potential targets for the signaling pathway and tumor cell–specific inhibitors.

Molecular Profiling Associated with Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2)-Mediated Carcinogenesis in Gastric Cancer.

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. We showed previously that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), a serine-threonine kinase, is highly expressed in gastric cancer and leads to progression. In the present study, we identified the molecular networks involved in CAMKK2-mediated progression of gastric adenocarcinoma. Treatment of gastric cancer cell lines with a CAMKK2 inhibitor, STO-609, resulted in decreased cell migration, invasion, and colony-forming ability and a G1/S-phase arrest. In addition, tandem mass tag (TMT)-based quantitative proteomic analysis resulted in the identification of 7609 proteins, of which 219 proteins were found to be overexpressed and 718 downregulated (1.5-fold). Our data identified several key downregulated proteins involved in cell division and cell proliferation, which included DNA replication licensing factors, replication factor C, origin recognition complex, replication protein A and GINS, and mesenchymal markers, upon CAMKK2 inhibition. Immunoblotting and immunofluorescence results showed concordance with our mass spectroscopy data. Taken together, our study supports CAMKK2 as a novel therapeutic target in gastric cancer.