Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is well known that point mutations exist in oncogenes and tumor suppressor genes of tumor cells, and one of the causes of these mutations may be misincorporation by error-prone DNA polymerases. This hypothesis is supported by the observation of decreased fidelity levels of DNA polymerases in mouse spleen containing tumorigenic cells after infection with Friend virus, and in aged animals that suffer high rates of tumorigenesis. However, this decrease in fidelity is disadvantageous for tumor cells maintained by serial transplantation. Therefore, we measured the fidelity levels of DNA polymerases in tumor cells transplanted through many passages. The fidelity levels of DNA polymerases from Yoshida ascites hepatoma, Rhodamine sarcoma, mouse ascites hepatoma-134, and Ehrlich ascites carcinoma cells derived from rats and mice are very high for in-vitro DNA synthesis on synthetic polynucleotides. These results suggest that many kinds of mutant cells arise during tumorigenesis. Among these mutant cells, cells showing decreased DNA polymerase(s) fidelities are present and these cells may undergo cell death. On the other hand, cells with mutations in various oncogenes and tumor suppressor genes and without mutations in DNA polymerase genes may survive as serially transplantable tumor cells.
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PMID:Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells. 988 76

The ends of human chromosomes (telomeres) consist of tandem repeats of the sequence TTAGGG. Telomeres lose up to 200 base pairs of DNA per cell division due to the inability of DNA polymerase to completely replicate the chromosomal ends. Chromosomal shortening ultimately leads to senescence and cell death in normal cells. However, some immortal cells do not lose telomeric sequence during DNA replication. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as a template. To elucidate potential mechanisms for telomerase regulation, we tested human squamous cell carcinoma lines (SCCs) for telomerase activity. All SCC lines expressed high levels of telomerase activity. Synchronization in specific cell cycle phases caused marked reduction in telomerase activity in G0 and S, but not in G1 or M. Reduction in telomerase activity correlated with induction of Rb protein in these phases. Overexpression of full length Rb resulted in significant downregulation of telomerase activity. However, expression of an Rb N-terminal oligomerization domain deletion construct, a C-terminal DNA binding domain deletion construct, or a pocket domain mutant failed to downregulate telomerase activity. We concluded that functionally intact Rb was required for cell cycle-dependent downregulation of telomerase activity in SCC lines.
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PMID:Intact functional domains of the retinoblastoma gene product (pRb) are required for downregulation of telomerase activity. 1032 Jul 73

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.
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PMID:The BFRF1 gene of Epstein-Barr virus encodes a novel protein. 1070 40

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange. In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins.
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PMID:Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers. 1076 Aug 17

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.
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PMID:p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase. 1087 24

The carcinogenic plant extract aristolochic acid (AA) is thought to be the major causative agent in the development of urothelial carcinomas found in patients with Chinese herb nephropathy (CHN). These carcinomas are associated with overexpression of p53, suggesting that the p53 gene is mutated in CHN-associated urothelial malignancy. To investigate the relation between AA-DNA adduct formation and possible p53 mutations, we mapped the distribution of DNA adducts formed by the two main components of AA, aristolochic acid I (AAI) and aristolochic acid II (AAII) at single nucleotide resolution in exons 5-8 of the human p53 gene in genomic DNA. To this end, an adduct-specific polymerase arrest assay combined with a terminal transferase-dependent PCR (TD-PCR) was used to amplify DNA fragments. AAI and AAII were reacted with human mammary carcinoma (MCF-7) DNA in vitro and the major DNA adducts formed were identified by the (32)P-postlabeling method. These adducted DNAs were used as templates for TD-PCR. Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra thus obtained showed a preference for reaction with purine bases in the human p53 gene for both activated compounds. For both AAs, adduct distribution was not random; the strongest signals were seen at codons 156, 158-159 and 166-167 for exon 5, at codons 196, 198-199, 202, 209, 214-215 and 220 for exon 6, at codons 234-235, 236-237 and 248-249 for exon 7 and at codons 283-284 and 290-291 for exon 8. Overall guanines at CpG sites in the p53 gene that correspond to mutational hotspots observed in many human cancers seem not to be preferential targets for AAI or II. We compared the AA-DNA binding spectrum in the p53 gene with the p53 mutational spectrum of urothelial carcinomas found in the human mutation database. No particular pattern of polymerase arrest was found that predicts AA-specific mutational hotspots in urothelial tumors of the current p53 database. Thus, AA is not a likely cause of non-CHN-related urothelial tumors.
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PMID:Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. 1115 51

We assessed the status of P53 in 32 surgically treated non-small cell lung cancers (NSCLC) by using yeast functional assay. For functional assay, total RNA extracted from fresh-frozen specimens was reverse transcribed and P53 cDNAs were PCR-amplified using Pfu DNA polymerase (Stratagene). The transcriptional competence of the P53 cDNA was then tested in a yeast reporter strain. 20 of the 32 (69%) NSCLC patients contained mutant P53 in the yeast functional assay with the higher frequency in squamous cell carcinoma (14/17, 82%) than in adenocarcinoma (5/10, 50%) and large cell carcinoma (3/5, 60%) (p<0.01, chi2 test). No significant difference was observed with respect to the TNM. Preliminary survival analysis showed that patients scoring positive for the yeast test had shorter disease-free survival (median = 10 months) than those that scored negative (median > 21 months). Our results suggest that yeast functional assay is not only an improved method to examine the status of P53, but might hopefully improve understanding of the role of mutant P53 in the clinical evaluation of NSCLC.
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PMID:Detection of P53 abnormalities in non-small cell lung cancer by yeast functional assay. 1137 99

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations of genes encoding for proteins of the mismatch repair (MMR) machinery. The majority of mutations occur in the MLH1 and MSH2 genes, and consist of splice-site, frameshift and nonsense changes, leading to loss of protein function. In this study, we screened 7 HNPCC families for MLH1/MSH2 mutations. Sequence changes were identified in 5 families. Four alterations were novel 1- or 2-bp deletions or insertions causing a frameshift and appearance of premature stop codons (MLH1: c.597-598delGA, c.1520-1521insT; MSH2: c.1444delA, c.119delG). The four small insertions/ deletions were located within stretches of simple repeated sequences. By reviewing the HNPCC mutation database, we found that the majority of 1-2 bp frameshift mutations similarly affects simple repetitive stretches, pointing to DNA polymerase slippage during replication as the most likely source of such errors. We also evaluated microsatellite instability (MSI) in a breast carcinoma (BC) from an MLH1 mutation carrier. While a colon cancer from the same individual showed MSI, the BC specimen was MSI-negative, indicating that development of the latter tumor was unrelated to MMR impairment, despite presence of a constitutional MLH1 mutation. Hum Mutat 17:521, 2001.
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PMID:Four novel MSH2 and MLH1 frameshift mutations and occurrence of a breast cancer phenocopy in hereditary nonpolyposis colorectal cancer. 1138 12

MCF 7 (human breast carcinoma cells) and mutants transfected with the DNA polymerase beta gene were tested for response to cisplatin, radiation and combined treatments. The transfected cells showed a higher level of polymerase beta activity and were more resistant to radiation and cisplatin compared to the parental cell line. Further studies showed that for isosurvival treatments the mutant cells were more effective in sublethal radiation damage repair compared to the parental line. The combination of cisplatin with radiation showed effective radiosensitization which was less in the mutants compared to the parental line. In addition, the sequence of cisplatin before irradiation was more effective then cisplabn after irradiation. Pre-exposure to low levels of cisplatin for up to 24 h before irradiation showed a small significant adaptive response in one mutant line at 8 h and while similar trends were observed in the parental lines at earlier times they were not significant. In summary our data show that polymerase beta and thus base excision repair may play a role in cellular responses to cisplatin and radiation.
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PMID:The response of human breast tumour cell lines with altered polymerase beta levels to cisplatin and radiation. 1149 1

Using anti-p53 (PAb1801 and PAb240), anti-DNA polymerase alpha and Ki-67 monoclonal antibodies, the expression of p53 was studied in 11 normal endometria, 14 endometrial hyperplasias and 27 endometrial carcinomas and its relationship to the proliferative activity of the tumors was examined. Normal endometria and simple hyperplasias were completely negative for p53. The PAb1801 indices of complex hyperplasias and complex atypical hyperplasias were 2.5+/-1.8% and 5.0+/-3.2%, respectively. The PAb1801 indices of grade 1, grade 2 and grade 3 endometrial carcinomas were 10.2+/-14.2%, 44.4+/-29/0% and 45.0+/-32.5%, respectively. These results indicate a progressively enhanced p53 expression in the sequence from normal endometrium, through hyperplasia to carcinoma. A significant correlation between p53 expression and labeling indices of Ki-67 and DNA polymerase alpha was observed in endometrial carcinomas. The endo-metrial carcinomas with p53 overexpression developed mainly in post-menopausal patients and were frequently high-grade tumors with deep myometrial invasion. These findings may indicate that overexpression of p53 protein contributes to the proliferative activity of the tumor cells.
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PMID:Immunohistochemical study of p53 expression in endometrial carcinomas: correlation with markers of proliferating cells and clinicopathologic features. 1157 70


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