Causal genetic regulation of DNA replication on immune microenvironment in colorectal tumorigenesis: Evidenced by an integrated approach of trans-omics and GWAS

The interplay between DNA replication stress and immune microenvironment alterations is known to play a crucial role in colorectal tumorigenesis,but a comprehensive understanding of their association with and relevant biomarkers involved in colorectal tumorigenesis is lacking.To address this gap,we conducted a study aiming to investigate this association and identify relevant biomarkers.We analyzed transcriptomic and proteomic profiles of 904 colorectal tumor tissues and 342 normal tissues to examine pathway enrichment,biological activity,and the immune microenvironment.Additionally,we evaluated genetic effects of single variants and genes on colorectal cancer susceptibility using data from genome-wide association studies(GWASs)involving both East Asian(7062 cases and 195745 controls)and European(24476 cases and 23073 controls)populations.We employed mediation analysis to infer the causal pathway,and applied multiplex immunofluorescence to visualize colocalized biomarkers in colorectal tumors and immune cells.Our findings revealed that both DNA replication activity and the flap structure-specific endonuclease 1(FEN1)gene were significantly enriched in colorectal tumor tissues,compared with normal tissues.Moreover,a genetic variant rs4246215 G>T in FEN1 was associated with a decreased risk of colorectal cancer(odds ratio=0.94,95%confidence interval:0.90–0.97,P_(meta)=4.70×10^(-9)).Importantly,we identified basophils and eosinophils that both exhibited a significantly decreased infiltration in colorectal tumors,and were regulated by rs4246215 through causal pathways involving both FEN1 and DNA replication.In conclusion,this trans-omics incorporating GWAS data provides insights into a plausible pathway connecting DNA replication and immunity,expanding biological knowledge of colorectal tumorigenesis and therapeutic targets.

Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB),which leads to considerable crop losses.Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously,we demonstrated that the S.turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.However,how the regulatory mechanism works was still largely unknown.Here,we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.We performed RNA-Seq analysis to identify S.tuicica genes responsive to DNA replication stress.In the current work,we found that appressorium-mediated maize infection by S.turcica is significantly blocked by S-phase checkpoint.A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S.turcica during the replication stress.The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain,multicopper oxidase,ABCtransporter families,cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.In addition,we demonstrated that autophagy in S.turcica is up-regulated by ATR as a defense response to stress.We identified StATG3,StATG4,StATG5,StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.We therefore propose that in response to genotoxic stress,S.turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection,and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection,aside from the more conservative responses in eukaryotes.

Artesunate Reduces Proliferation, Interferes DNA Replication and Cell Cycle and Enhances Apoptosis in Vascular Smooth Muscle Cells

Summary: This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded. The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated. DNA replication was evaluated by [3H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G 0/G 1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G 0/G 1 phase.

Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves.The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor.Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata.Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor(tricyclazole,TCZ),a DNA replication inhibitor(hydroxyurea,HU),and an autophagy inhibitor(3-methyladenine,3-MA),on appressorium turgor and glycerol content.As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.Glycogen and liposomes are the main substances producing glycerol.It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development.This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection.

Acceleration of DNA Replication of Klenow Fragment by Small Resisting Force

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force,attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.

Essential functions of iron-requiring proteins n DNA replication, repair and cell cycle contro

Eukaryotic cells contain numerous iron-requiring pro- teins such as iron-sulfur （Fe-S） cluster proteins, hemoproteins and ribonucleotide reductases （RNRs）. These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these iron- requiring proteins and is genetically associated with diseases characterized by DNA repair defects in mam- mals. Organisms have evolved multi-layered mecha- nisms to regulate iron balance to ensure genome stability and cell development. This review briefly pro- vides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.

Demonstration of biophoton-driven DNA replication via gold nanoparticle-distance modulated yield oscillation

Biologically,there exist two kinds of syntheses:photosynthesis and ATP-driven biosynthesis.The light harvesting of photosynthesis is known to achieve an efficiency of〜95%by the quantum energy transfer of photons.However,how the ATP-driven biosynthesis reaches its high efficiency still remains unknown.Deoxynucleotide triphosphates(dNTPs)in polymerase chain reaction(PCR)adopt the identical way of ATP to release their energy,and thus can be employed to explore the ATP energy process.Here,using a gold nanoparticle(AuNP)enhanced PCR(AuNP-PCR),we demonstrate that the energy released by phosphoanhydride-bond(PB)hydrolysis of dNTPs is in form of photons(PB-photons)to drive DNA replication,by modulating their resonance with the average inter-AuNP distance(D).The experimental results show that both the efficiency and yield of PCR periodically oscillate with D increasing,indicating a quantized process,but not simply a thermal one.The PB-photon wavelength is further determined to 8.4 pm.All these results support that the release,transfer and utilization of bioenergy are in the form of photons.Our findings of ATP-energy quantum conversion will open a new avenue to the studies of high-efficiency bioenergy utilization,biochemistry,biological quantum physics,and even brain sciences.

A Combined Computational and Experimental Study on the Structure-Regulation Relationships of Putative Mammalian DNA Replication Initiator GINS

GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized. In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS mRNA levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17β-Estradiolstimulated estrogen receptor a, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

HPV18 DNA replication inactivates the early promoter P_(55) activity and prevents viral E6 expression

Dear Editor,Human papillomaviruses(HPV)are a large group(>200genotypes)of small double-stranded DNA viruses(https://pave.niaid.nih.gov/).Although infections by most HPV types are asymptomatic,persistent infections in cervical and ano-genital epithelia by high-risk

5-Formyluracil targeted biochemical reactions with proteins inhibit DNA replication,induce mutations and interference gene expression in living cells

Covalent DNA–protein cross-links are toxic DNA lesions that interfere with essential biological processes,which can cause serious biological consequences,such as genomic instability and protein misexpression.5-Formyluracil(5 fU) as an important modification in DNA,which is mainly from oxidative damage,exists in a variety of cells and tissues.We have reported that 5 fU mediated DNA–protein conjugates could exist in human cells [Zhou et al.CCS Chem.2(2020) 54–63].We now aimed to explore its potential biological effects in vitro and in vivo.In this paper,we firstly reported that 5 fU intermediated DNA–peptide or DNA–protein conjugates(both were called DPCs) could inhibit different polymerases bypass or cause mutations.Then we further investigated the functional impacts caused by 5 fU-mediated DPCs,which appeared in different gene expression components [in the promoter sequence or 50-untranslated regions(UTR)].These results together may contribute to a broader understanding of DNA–protein interactions as well as the biological functions associated with 5 fU.

Next-generation sequencing-based analysis of the effect of N^(6)-methyldeoxyadenosine modification on DNA replication in human cells

N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.

DNA replication components as regulators of epigenetic inheritance---lesson from fission yeast centromere

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stabil- ity. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

DNA replication licensing control and rereplication prevention

Eukaryotic DNA replication is tightly restricted to onlyonce per cell cycle in order to maintain genome stability.Cells use multiple mechanisms to control the assemblyof the prereplication complex (pre-RC), a process knownas replication licensing. This review focuses on theregulation of replication licensing by posttranslationalmodifications of the licensing factors, including phosphorylation, ubiquitylation and acetylation. These modifications are critical in establishing the pre-RCcomplexes as well as preventing rereplication in each cellcycle. The relationship between rereplication and diseases, including cancer and virus infection, is discussedas well.

RPA functions as a platform for coupling of nucleosome assembly to DNA replication

Subject Code:C05 With the support by the National Natural Science Foundation of China,the research team led by Dr.Li Qing(李晴)at the State Key Laboratory of Protein and Plant Gene Research,School of Life Sciences and Peking-Tsinghua Center for Life Sciences,Peking University,Beijing,recently reported that

New insights into ATR inhibition in muscle invasive bladder cancer:The role of apolipoprotein B mRNA editing catalytic subunit 3B

Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Novel coumarone-derived(S,E)-4-(4-fluorobenzylidene)-3-phenylchroman-3-ol inhibits muscle-invasive bladder cancer cells by repressing the S and G2 cell cycle phases

Background:This study aimed to select compounds with unique inhibitory effects on muscle-invasive bladder cancer(MIBC)from coumarone derivatives with similar parent nuclear structures and to reveal their tumor-suppressive effects using various approaches.Methods:Bladder cancer cell lines SW780 and T24,as well as human normal bladder epithelial cell line SV-HUC-1 were selected as the study model,and these urinary system cells were co-incubated with various concentrations of(S,E)-4-(4-methylbenzylidene)-3-phenylchroman-3-ol,(S,E)-4-(4-isocyanobenzylidene)-3-phenylchroman-3-ol,(S,E)-4-(4-fluorobenzylidene)-3-phenylchroman-3-ol(FPO),and(S,E)-3-phenyl-4-(4-(trifluoromethoxy)benzylidene)chroman-3-ol.Cell activity was detected using cell counting kit-8.FPO showed the strongest inhibitory effect on MIBC cells;therefore,it was selected for further experiments.We monitored the FPO-induced T24 cell morphological changes with an inverted microscope.The FPO-inhibited migration of T24 cells was examined using a cell scratch assay.We detected the clonogenic ability of T24 cells through a clone formation test and evaluated their proliferative ability using a 5-ethynyl-2’-deoxyuridine fluorescence staining kit.The inhibitory effect of FPO against the cell cycle was monitored using flow cytometry,and its suppressive effect on the DNA replication ability of T24 cells was detected using double fluorescence staining(Ki67 and phalloidin).Results:Among the four candidate coumarone derivatives,FPO showed the most significant inhibitory effect on MIBC cells and was less toxic to normal urothelial cells.FPO inhibited T24 cell growth in time and dose-dependent manners(the half-inhibitory concentration is 8μM).FPO significantly repressed the proliferation,migration,and clonogenic ability of bladder cancer T24 cells.Cell mobility was significantly inhibited by FPO:30μM FPO almost completely repressed migration occurred at after 24 h treatment.Moreover,FPO significantly suppressed the clonogenicity of bladder cancer cells in a dose-dependent manner.Mechanistically,FPO targeted the cell cycle,arresting the S and G2 phases on bladder cancer T24 cells.Conclusion:We discovered a novel anticancer chemical,FPO,and proposed a potential mechanism,through which it suppresses MIBC T24 cells by repressing the cell cycle in the S and G2 phases.This study contributes to the development of novel anticancer drugs for MIBC.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

Lunasin peptide promotes lysosome-mitochondrial mediated apoptosis and mitotic termination in MDA-MB-231 cells

Lunasin,a novel bioactive peptide,is well-known for its anti-proliferation activity.However,the mechanism of this effect is still poorly reported.Here,synthesized lunasin was used and its anti-proliferative function was observed at the concentration of 0.25 mg/m L in human breast cancer cell MDA-MB-231.Conjoint analysis of transcriptome and proteome of MDA-MB-231 cells was further performed.The results demonstrated that cysteinyl aspartate specific proteinase(CASP)3,CASP 7,and CASP 14 were significantly up-regulated after lunasin exposure,together with an increased Bax/Bcl-2 ratio from 22.9 to 210.6,which indicated that caspase-mediated mitochondria intrinsic apoptosis was highly activated.Moreover,lysosomal pathway was signifi cantly suppressed under lunasin exposure,suggesting that lysosome may cooperate with mitochondria to participate in apoptosis.In addition,lunasin also down-regulated genes involved in DNA replication in MDA-MB-231 cells.Overall,our study reveals that the anti-proliferation effect of lunasin peptide might be triggered via the inhibition of DNA replication and cell mitosis,as well as the promotion of lysosome-mitochondrial mediated cell apoptosis.

Study on the Relationship between Cytogenetics and Phenotypic Effect in Turner＇s Syndrome

The cytogenetics and clinical stigmata in 5 cases of Turner's syndrome were studied. Three of them were non-mosaic i(Xq) and two with partial monosome of a X chromosome short are (Xp21), whose DNA replication patterns of inactive X chromosome were analyzed by RBG technique. Results showed that differences between the replication patterns in cases of X chromosome deletion (Xp21) and normal females existed; that the behavior of abnormal X expressed nonrandom inactivation. It was suggested that the phenotype may be closely related with both X chromosome replication pattern and its inactivation behavior,which might be useful in genetic counselling.

Discovery of a potent and selective cell division cycle 7 inhibitor from 6-(3-fluoropyridin4-yl)thieno[3,2-d]pyrimidin-4(3H)-one derivatives as an orally active antitumor agent

To the Editor:Kinase cell division cycle 7(CDC7),a cell division cycle protein,takes a vital role in mediating DNA replication1.CDC7 complexes in the nucleus can phosphorylate the minichromosome maintenance complex(MCM)family members that bind to chromosomes.In addition,CDC7 kinase,as a molecular switch regulating DNA replication,can mediate DNA damage signaling pathways to stimulate cell cycle termination as well as DNA replication2.Studies have shown that CDC7 is overexpressed in many types of cancer cells,and its overexpression was related to poor patient survival,tumor grade,genetic instability,aneuploidy and so on3.Therefore,CDC7 is a promising target for antitumor therapy.