Systematic analysis of DNA polymerases as therapeutic targets in pan-cancers

Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy targets are not well understood.Methods:We conducted a systematic analysis using TCGA Pan-Cancer Atlas data and Gene Set Cancer Analysis results to examine the expression profiles of 15 DNA polymerases(POLYs)and their clinical correlations.We also evaluated the prognostic value of POLYs by analyzing their expression levels in relation to overall survival time(OS)using Kaplan-Meier survival curves.Additionally,we investigated the correlations between POLY expression and immune cells,DNA damage repair(DDR)pathways,and ubiquitination.Drug sensitivity analysis was performed to assess the relationship between POLY expression and drug response.Results:Our analysis revealed that 14 out of 15 POLYs exhibited significantly distinct expression patterns between tumor and normal samples across most cancer types,except for DNA nucleotidylexotransferase(DNTT).Specifically,POLD1 and POLE showed elevated expression in almost all cancers,while POLQ exhibited high expression levels in all cancer types.Some POLYs showed heightened expression in specific cancer subtypes,while others exhibited low expression.Kaplan-Meier survival curves demonstrated significant prognostic value of POLYs in multiple cancers,including PAAD,KIRC,and ACC.Cox analysis further validated these findings.Alteration patterns of POLYs varied significantly among different cancer types and were associated with poorer survival outcomes.Significant correlations were observed between the expression of POLY members and immune cells,DDR pathways,and ubiquitination.Drug sensitivity analysis indicated an inverse relationship between POLY expression and drug response.Conclusion:Our comprehensive study highlights the significant role of POLYs in cancer development and identifies them as promising prognostic and immunological biomarkers for various cancer types.Additionally,targeting POLYs therapeutically holds promise for tumor immunotherapy.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

中国蓑鲉亚科鱼类DNA条形码及分子系统进化研究

为从分子水平分析我国蓑鲉亚科鱼类分类关系,通过PCR扩增及测序获得了我国8种蓑鲉亚科鱼类COⅠ基因部分序列,结合GenBank下载的6种蓑鲉,共计14种40个个体线粒体COⅠ基因序列,利用MEGA X计算了序列信息和遗传距离,利用基于最大似然法和邻接法构建分子系统进化树。结果显示:除盔蓑鲉(Ebosia bleekeri)和安狭蓑鲉(Pteroidichthys amboinensis)只有一个样本无法测定外,其余12种蓑鲉亚科鱼类的种间平均遗传距离是种内平均遗传距离的69.17倍,显示COⅠ基因可作为蓑鲉亚科鱼类的有效DNA条形码基因。进化树上,蓑鲉亚科部分属并未能聚成属间单系,其中蓑鲉属(Pterois)与短鳍蓑鲉属(Dendrochirus)在进化树上形成多个分支。在蓑鲉属中,翱翔蓑鲉(P.volitans)与斑鳍蓑鲉(P.miles)聚为一支,两者关系较近,但COⅠ遗传距离为0.042,大于Hebert推荐的0.020作为最小区分物种的遗传距离,支持两者为两个独立物种的观点;触须蓑鲉(P.antennata)、辐蓑鲉(P.radiate)和黑颊蓑鲉(P.mombasae)聚为一支,独立于翱翔蓑鲉与斑鳍蓑鲉的分支之外,并与短鳍蓑鲉属(Dendrochirus)的花斑短鳍蓑鲉(D.zebra)以及盔蓑鲉属(Ebosia)的种类聚在一起,与近期有关蓑鲉亚科各种属分子聚类研究结果相似;但这3种蓑鲉是否可以从蓑鲉属中独立出来归为Pteropterus属,还需要后续更多的蓑鲉亚科鱼类样品研究验证。

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

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The characteristics of DNA repair synthesis induced by DNA polymerase β in hepatoma cells after γ rays irradiation

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基于COⅠ基因的5种新疆土著鱼类DNA条形码鉴定及聚类分析

为探讨DNA条形码技术在新疆土著鱼类物种鉴定中的有效性并保护本土渔业重要种质资源,本研究采用DNA条形码技术对裂腹鱼亚科(Schizothoracinae)和高原鳅属(Triplophysa)鱼类进行COⅠ基因序列扩增,并与NCBI数据库进行序列比对,结合遗传距离和系统发育树进行物种鉴定分析。结果表明,33尾个体分属4属5种,其中有13个单倍体;COⅠ基因序列中(G+C) mol%值为46.7%,远小于(A+T) mol%值53.3%,表现出明显的反G偏倚,第三位密码子处的(G+C) mol%值为55.9%,远高于第一和第二密码子处的(G+C) mol%值,R值(转换/颠换)为2.10;种间平均遗传距离(11.55±0.95)%,为种内平均、遗传距离(0.14±0.10)%的43.8倍,远远大于Hebert所提出的“10倍法则”;系统发育树表明,裂腹鱼亚科和高原鳅属鱼类分为两支,其中,裂腹鱼亚科的塔里木裂腹鱼(Schizothorax biddulphi)、宽口裂腹鱼(Schizothorax eurystomus)、扁吻鱼(Aspiorhynchus laticeps)、斑重唇鱼(Diptychus maculatus)的同种不同个体各自独立聚为一支,显示出明显的单系性;高原鳅属中的长身高原鳅(Triplophysa teunis)也独自立为一支,所有物种都被区分开。综上,基于COⅠ基因的DNA条形码技术可以有效鉴定新疆5种土著鱼类。

Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone （ranged from 10 μmol/L to 120 μmol/L） for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis （SCGE）] were performed respectively to detect the toxicity of hydroquinone. Results assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

在他们描述脱氧核糖核酸双螺旋，沃森和克里克的结构写了什么可以是在科学文学的最大的保守的陈述之一的精液的出版，也就是那“这没疏忽特定的配对我们立即要求了为遗传物质建议可能的拷贝机制。“半个世纪以后，我们更充分欣赏它是与精确性复制 60 亿核苷酸的巨大的挑战需要稳定地在许多代上维持人的染色体的。这挑战也许比大 50 年以前被认识到，因为随后的研究表明了染色体能被在脱氧核糖核酸而且由现在质问到复制的正常脱氧核糖核酸的各种各样的顺序主题产生潜在地细胞毒素、诱变的损害的一个大数字和变化的环境压力使动摇不仅。向染色体稳定性的许多决定因素的更好的理解，这章考察 undamaged 和损坏脱氧核糖核酸与被拷贝的忠实，与真核细胞的 B 家庭和 Y 家庭 DNA 的一个焦点，并且考虑这忠实怎么被完成。

Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-homologous DNA end joining： relevance to cancer, aging, and the immune system

加入的 Nonhomologous 脱氧核糖核酸结束(NHEJ ) 是为在人的房间并且在多细胞的优核质的双海滨脱氧核糖核酸裂缝的修理的主要小径。双海滨裂缝的原因经常碎裂在损坏的地点的脱氧核糖核酸，导致在那里的信息的损失。NHEJ 不恢复失去的信息并且可以将切除另外的核苷酸在修理过程期间。修理大量 overhang 并且损坏配置的能力反映 NHEJ 的核酸酶，聚合酶，和连接酶的灵活性。单个部件的灵活性也解释 NHEJ 能在修理脱氧核糖核酸结束的任何给定的对的方法的大数字。局部地，在 NHEJ 的地点，修理可以贡献癌症和老化的信息的损失，而是由 NHEJ 的行动保证染色体的全部片断没被失去。

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Mutation of DNA polymerase p in esophageal carcinoma of different regions

AIM: To observe the variation of DNA polymerase β (polβ)in esophageal carcinoma.METHODS: Thirty specimens containing adjacent normal epithelial tissues were collected from patients in Linzhou region (a high risk area for esophageal squamous carcinoma) and 25 specimens were from a non-high risk area. Total RNA was extracted from the samples and reverse transcription polymerase chain reaction (RT-PCR) was performed. PCR products were cloned and sequenced to investigate the polβ gene with DNASIS and OMIGA. Statistical significance was evaluated using the x2 test.RESULTS: High-incidence area group: polβ gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30corresponding adjacent normal tissue specimens. Non high-incidence area group: polβ gene variation was detected in 5 of 25 esophageal carcinoma tissue specimens,and no variation was found in 25 corresponding adjacent normal tissue specimens. The incidence of polβ gene variation observed in the high-incidence area group was significantly higher than in the non-high incidence area group. Two mutation hot spots (454-466 and 648-670 nt)and a 58 bp deletion (177-234 nt) were found.CONCLUSION: Variations of polβ perform different functions between the high-incidence areas and the other areas, and may play a more important role in the high-incidence areas.

YMDD variants of HBV DNA polymerase gene: Rapid detection and clinicopathoiogical analysis with long-term Iamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants.METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene.RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT.CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.

CONFORMATION STUDY OF THE LARGE FRAGMENT OF E. COLI DNA POLYMERASE I BY STM

The large fragment of E.coli DNA polymerase I is imaged by scanning tunneling microscope. The specimen is deposited on highly oriented pyrolytic graphite surface, and then covered with pure paraffin oil in order to maintain hydration of the molecules. Images of the enzyme reveal an ellipsoid shape of 5.5-6.0nm wide and 7.0 -7.5 nm long. The conformation of the enzyme is in agreement with the model derived from X- ray crystallography studies.

SELEX技术中制备单链DNA的方法

制备单链DNA(ssDNA)是许多常用实验中的重要内容,是通过指数富集的配体系统(SELEX)进行体外筛选核酸适配体的关键步骤之一。目前已报道多种方式可通过双链DNA(dsDNA)制备ssDNA,包括链霉亲和素包被磁珠分离、不对称PCR、不等大小引物PCR、酶消化、不对称PCR结合酶消化和不对称乳液PCR等,而如何快速高效地制备ssDNA是研究重点。本文综述了常用的几种通过聚合酶链式反应(PCR)制备ssDNA的方法,对其进行综合分析。根据产物的纯度、操作难易、实验成本等方面综合考虑,选择产物高纯度、操作简便且实验成本较低的ssDNA制备方法,为具有不同需求的应用场景提供参考依据。

Detection of hepatitis B viral DNA in liver with polymerase chain reaction

The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.

THE RADIOBIOLOGIC CHARACTERISTICS OF DNA POLYMERASE β IN HEPATOMAS

To investigate the effects of γ rays on DNA polymerase β properties and its DNA repair functions before or after γ rays exposure, DNA polymerase βactivity, gene expression and mRNA levels in SMMC-LTNM hepatomas horn on nude mice or the samples of the liver cancer tissues from 15 patients were measured with 3H-TTP incorporation test, immunocytochemistry and cytoplasmic dot hybridization analysis, respectively.Irradiation was carried out with 60Co-γ rays at ice bath. It was found that DNA polymerase β activity, gene expression and the amount of mRNA were much higher in hepatoma cells than those in normal hepatocytes (P<0.01). In vitro studies, the enzyme activity both in hepatoma and normal liver cells appeared unchanged within 40 Gy γ-ray exposure. Following whole-body exposure of the nude mice bearing SMMC-LTNM with 2 Gy or 4 Gy of γ rays, DNA polymerase β activity in hepatoma increased temorarily at 48 hours postirradiation, and its gene expression seemed more active.The euzyme mRNA increased to 1.76-fold of the control group. 72 hours after exposure, all of these changes returned to normal levels. DNA polymerase βparticipated in DNA repair synthesis and this effect was different between hepatoma and hepatocytes because there were some biologic differences of the enzyme between hepatoma cells and normal liver cells. These data suggested that DNA polymeraseβactivity, its gene expression and mRNA level in hepatomas could increased temporarily after γ rays exposure, which may facilitate the cells to repair DNA damages from radiation.

Roles of DNA polymerase β on repair of DNA damaged by γ－rays irradiation

The roles of DNA polymerase β in the repair synthesis of γ-rays irradiated DNA from calf thymus .SMMC-LTNM hepatoma and nude mouse hepatocytes were evaluated, using NEM or d2TTP as selective inhibitors to DNA polymerase a or β . It was observed that the rate of [3H]-TTP incorporating into calf thymus DNA damaged by 10 Gy γ-ray was much higher( P<0.01 ) than that of the non-irradiated one. when there was some amount of recombinant rat DNA polymerase β in the reaction mixture. We also found that the [3H]-TTP incorporation rate reflecting the DNA repair synthesis increased gradually as γ-rays absorbed doserose to 20 Gy for hepatocyte nuclei or to 5 Gy for hepatoma nuclei. and then decreased slowly as the absorbeddose rose higher. These results suggest that DNA polyrnerase β could participate in DNA repair synthesis inboth normal and neoplastic nuclei irradiated by γ-rays and its roles in DNA repair may be related to cell typesand absorbed dose.

THE INHIBITORY EFFECT OF EXTRACT OF CAMELLIA SINENSIS AND EXTRACT OF CAMELLIA PTILOPHYLLA CHANG ON DNA POLYMERASE OF EHRLICH ASCITES CARCINOMA CELLS

Objective: To detect the effect of extract of Camellia Sinensis (ECS) and extract of Camellia Ptilophylla Chang (ECPC) on DNA polymerase (Pol) of Ehrlich ascites tumor cells Methods: Referring to the method of K Ono, Pol was extracted from Ehrlich ascites tumor cells in mice Pol α, β, and γ were separated by phosphocellulose column chromatography and were identified The effect of ECPC and ECS on Pol was studied Results: ECPC and ECS were shown to inhibit the activity of Pol α, β, and γ IC 50 values of ECS on Pol α , β, and γ were 10 2μg/ml, 9 9μg/ml and 28 9μg/ml respectively IC 50 values of ECPC on Pol α, Pol β and Pol γ were 5 6μg/ml, 15μg/ml and 14 7μg/ml respectively The modes of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA The Ki values of ECPC on Pol α , β, and γ were 2 68±0 12μg/ml, 2 24±0 12μg/ml , 2 56±0 18μg/ml Conclusion: ECPC and ECS were shown to have inhibitory effect on DNA polymerase of tumor cells The mode of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA

Expression of Human Cytomegalovirus DNA Polymerase in Insect Cells Using Baculovirus Expression System：Purification and Biochemical Characterizations

Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr Ⅱ-EcoRI DNA fragment with intact HCMV pci gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pci gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pci gene. In fection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 108 cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3'-5'and 5'-3' exonuclease activities.This enzyme is also sensitive to phosphono acetate.