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 ...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.展开更多
DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbit...DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.展开更多
Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Metho...Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.展开更多
AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing techn...AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.展开更多
AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reactio...AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.展开更多
Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-...Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.展开更多
AIM To investigate the effects of DNA repair synthesis induced by DNA polymerase β in hepatoma cells after γ ray irradiation. METHODS Cell nuclei were prepared from SMMC LTNM hepatoma which is a transplanted hu...AIM To investigate the effects of DNA repair synthesis induced by DNA polymerase β in hepatoma cells after γ ray irradiation. METHODS Cell nuclei were prepared from SMMC LTNM hepatoma which is a transplanted human liver cancer born on nude mice. Samples were irradiated with 60 Co γ rays at different doses or dose rates. N ethylmaleimide (NEM) and ddTTP were used as selective inhibitors to DNA polymerases. The reaction of DNA repair synthesis was carried out with the selective inhibitor test. RESULTS It was found that the 3H TTP incorporation in irradiated nuclei or calf thymus DNA was significantly higher than that in the non irradiated ones, under the conditions of DNA polymerase α or γ being inhibited. When NEM and ddTTP which selectively inhibits DNA polymerase β both existed in the DNA repair synthesis reaction mixture, the 3H TTP incorporation in irradiated DNA did not significantly increased. Furthermore, 3H TTP incorporation into DNA of SMMC LTNM hepatoma nuclei was higher than that of normal hepatocyte nuclei ( P <0 01). The DNA repair synthesis induced by DNA polymerase β reacted more fast in hepatoma nuclei than in hepatocyte nuclei. CONCLUSION The effects of DNA repair synthesis induced by DNA polymerase β in some tumor cells might be stronger than that in normal cells, which may facilitate the cells to repair DNA damages from radiation.展开更多
In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our...In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.展开更多
Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the sit...Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.展开更多
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. M...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.展开更多
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 r...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 po1β gene with DNASIS and OMIGA. Statistical significance was evaluated using the X^2 test. RESULTS： High-inddence area group： Polβ gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30 corresponding adjacent normal tissue specimens. Non high-incidence area group： po1β 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 po1β 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.展开更多
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) an...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.展开更多
BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breas...BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer.They are also effective for new and recurrent ovarian cancers that are BRCA-or homologous recombination deficiency(HRD)-positive.However,data on these mutations and PARPi use in the Middle East are limited.AIM To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer.METHODS This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations,and 25 of 65 ovarian cancer patients tested for HRD.These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023.Data were summarized using descriptive statistics and compared using counts and percentages.Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria.RESULTS Among the 472 breast cancer patients,12.1%underwent BRCA testing,and 38.5%of 65 ovarian cancer patients received HRD testing.Pathogenic mutations were found in 25.6%of the tested patients:26.3%breast cancers had germline BRCA(gBRCA)mutations and 24.0%ovarian cancers showed HRD.Notably,40.0%of gBRCA-positive breast cancers and 66.0%of HRD-positive ovarian cancers were Middle Eastern and Asian patients,respectively.PARPi treatment was used in 5(33.3%)gBRCA-positive breast cancer patients as first-line therapy(n=1;7-months progression-free),for maintenance(n=2;>15-months progression-free),or at later stages due to compliance issues(n=2).Four patients(66.6%)with HRD-positive ovarian cancer received PARPi and all remained progression-free.CONCLUSION Lower testing rates but higher BRCA mutations in breast cancer were found.Ethnicity reflected United Arab Emirates demographics,with breast cancer in Middle Eastern and ovarian cancer in Asian patients.展开更多
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 ma...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.展开更多
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 ex...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.展开更多
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 ho...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.展开更多
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 polyme...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.展开更多
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 ...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展开更多
基金supported by the project of funds by the Consultation of Provincial Department and University for S&T Innovation granted by Hebei Provincial Department of Science and Technology and Hebei Medical University(2020TXZH04).
文摘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.
文摘DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.
文摘Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.
文摘AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.
文摘AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.
文摘Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.
文摘AIM To investigate the effects of DNA repair synthesis induced by DNA polymerase β in hepatoma cells after γ ray irradiation. METHODS Cell nuclei were prepared from SMMC LTNM hepatoma which is a transplanted human liver cancer born on nude mice. Samples were irradiated with 60 Co γ rays at different doses or dose rates. N ethylmaleimide (NEM) and ddTTP were used as selective inhibitors to DNA polymerases. The reaction of DNA repair synthesis was carried out with the selective inhibitor test. RESULTS It was found that the 3H TTP incorporation in irradiated nuclei or calf thymus DNA was significantly higher than that in the non irradiated ones, under the conditions of DNA polymerase α or γ being inhibited. When NEM and ddTTP which selectively inhibits DNA polymerase β both existed in the DNA repair synthesis reaction mixture, the 3H TTP incorporation in irradiated DNA did not significantly increased. Furthermore, 3H TTP incorporation into DNA of SMMC LTNM hepatoma nuclei was higher than that of normal hepatocyte nuclei ( P <0 01). The DNA repair synthesis induced by DNA polymerase β reacted more fast in hepatoma nuclei than in hepatocyte nuclei. CONCLUSION The effects of DNA repair synthesis induced by DNA polymerase β in some tumor cells might be stronger than that in normal cells, which may facilitate the cells to repair DNA damages from radiation.
文摘In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.
文摘Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.
基金This work was supported by a grant from the Major State Basic Research Development Program of China (No. 2002CB512904, 2002CB512903).
文摘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.
基金Supported by the National Natural Science Foundation of China,No. 39870287
文摘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 po1β gene with DNASIS and OMIGA. Statistical significance was evaluated using the X^2 test. RESULTS： High-inddence area group： Polβ gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30 corresponding adjacent normal tissue specimens. Non high-incidence area group： po1β 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 po1β 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.
基金Supported by the National Natural Science Foundation of China, No. 30170363 the Major Science and Technology Program of Ministry of Education, No. 01003 the Major State Basic Research Development Program of China, No. 2002CB513100 the National Key Technology Research and Development Program of China during the 10~(th) Five-Year Plan Period, No. 2001BA703B05
文摘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.
文摘BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer.They are also effective for new and recurrent ovarian cancers that are BRCA-or homologous recombination deficiency(HRD)-positive.However,data on these mutations and PARPi use in the Middle East are limited.AIM To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer.METHODS This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations,and 25 of 65 ovarian cancer patients tested for HRD.These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023.Data were summarized using descriptive statistics and compared using counts and percentages.Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria.RESULTS Among the 472 breast cancer patients,12.1%underwent BRCA testing,and 38.5%of 65 ovarian cancer patients received HRD testing.Pathogenic mutations were found in 25.6%of the tested patients:26.3%breast cancers had germline BRCA(gBRCA)mutations and 24.0%ovarian cancers showed HRD.Notably,40.0%of gBRCA-positive breast cancers and 66.0%of HRD-positive ovarian cancers were Middle Eastern and Asian patients,respectively.PARPi treatment was used in 5(33.3%)gBRCA-positive breast cancer patients as first-line therapy(n=1;7-months progression-free),for maintenance(n=2;>15-months progression-free),or at later stages due to compliance issues(n=2).Four patients(66.6%)with HRD-positive ovarian cancer received PARPi and all remained progression-free.CONCLUSION Lower testing rates but higher BRCA mutations in breast cancer were found.Ethnicity reflected United Arab Emirates demographics,with breast cancer in Middle Eastern and ovarian cancer in Asian patients.
文摘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.
文摘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.
文摘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.
文摘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.
文摘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
