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)

6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A). LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer). When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.
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PMID:Mutagenic consequences of the incorporation of 6-thioguanine into DNA. 927 1

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH).
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PMID:The catalytic subunit of herpes simplex virus type 1 DNA polymerase contains a nuclear localization signal in the UL42-binding region. 1089 16

The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.
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PMID:Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor gene. 1095 64

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.
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PMID:Disruption of the developmentally regulated Rev3l gene causes embryonic lethality. 1105 Mar 92

An enhancer-like element VV16 from Vaccinia virus genome DNA was obtained by using the plasmid with CAT reporter gene. Sequence analysis showed the element of 112 bp is a part of the DNA-dependent RNA polymerase, polyA polymerase and DNA polymerase (RPO30 gene). It contains 4 AT-rich regions. Detection of beta-galactosidase activity showed that VV16 in the positive direction can increase the activity 9.0 times and VV16 in the negative direction can increase 4.1 times. The RNA dot blotting confirmed the enhancing activity of the element are on the transcription level. DNA deletion experiment indicated the sequences of 10 bp at the 5' end and 12 bp at the 3' end in the element are important to its function and the sequence from nt76 to nt82 is essential to its activity.
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PMID:[Functional and structural study of the prokaryotic enhancer-like element VV16 from vaccinia virus genome]. 1105 76

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.
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PMID:Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin. 1126 79

Directed evolution of Escherichia coli beta-galactosidase into variants featuring beta-glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3'-->5' exonuclease activity. beta-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F'128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F'128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.
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PMID:Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3'-->5' exonuclease activity of DNA polymerase III. 1128 11

Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.
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PMID:Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ. 1186 10

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.
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PMID:Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence. 1241 54

Topoisomerase I inhibitors have been shown to have clinical activity against human colorectal cancer. Previous studies showed that the cytotoxicity of camptothecin, a topoisomerase I inhibitor, occurs mainly in the S -phase of the cell cycle and is protectable by aphidicolin, an inhibitor of replicative DNA polymerase in some camptothecin-sensitive colorectal cells. Transcription factor E2F-1 regulates the G1/S transition, and recent studies have shown that E2F-1 potentiated the cytotoxicity of some cell-cycle-related drugs. Therefore, the present study was designed to investigate the effect of adenovirus-mediated E2F-1 gene transfer on chemosensitivity of colorectal cancer to camptothecin, in vitro and in vivo. Two human colorectal cancer cells, SW620 (mutant p53) and RKO (wild-type p53), were treated with camptothecin, alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ), or E2F-1 (Ad-E2F-1). E2F-1 overexpression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at low doses (less than the LD(20) dose) markedly increased the sensitivity of human colorectal cancer cells to camptothecin in vitro, which is because of induction of apoptosis. Aphidicolin did not have any protective effect on the Ad-E2F-1/camptothecin-mediated cytotoxicity. The level of topoisomerase I expression was not affected by combination treatment as well, suggesting that DNA replication and topoisomerase I activity may not account for the molecular mechanism of cell killing in response to Ad-E2F-1/camptothecin treatment. Fas and Fas ligand expression were not altered by treatment with camptothecin and/or Ad-E2F-1. Moreover, combination of camptothecin and Ad-E2F-1 has an additive antitumor effect in an in vivo nude mouse xenograft model. When combined with camptothecin, E2F-1 adenovirus therapy resulted in a 95.7% decrease in tumor size compared to control groups (P<.05). These results suggest a chemosensitization strategy that may have clinical utility in human colorectal cancer.
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PMID:E2F-1 overexpression sensitizes colorectal cancer cells to camptothecin. 1263 37


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