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)

We have established the first homologous cell-free DNA replication system for a papillomavirus. The replication of the human papillomavirus type 11 (HPV-11) origin was achieved by using human 293 cell extracts supplemented with the HPV-11 E1 and E2 proteins purified from insect cells infected with recombinant baculoviruses. Efficient replication depends on the HPV-11 origin, the HPV-11 E1 and E2 proteins, as well as human DNA polymerase alpha, delta, replication protein A, topoisomerase I, and topoisomerase II. High concentrations of E1 protein also promoted a low level of origin-independent replication which was suppressed by the addition of the E2 protein, whereas E2 protein stimulated origin-dependent replication. We also show that an intact E2 protein binding site was absolutely necessary for origin activity, as a strong HPV-11 origin was rendered inactive when one half-site of each of the three E2 binding sites was mutated. In contrast, there was only a relatively small reduction in this mutant origin activity when the cell extracts were supplemented with the bovine papillomavirus type 1 (BPV-1) proteins. These results suggest that the HPV-11 E2 protein plays a primary role in HPV origin recognition. Furthermore, unlike transient replication in which HPV-11 and BPV-1 viral proteins promote efficient replication of homologous and heterologous origins, efficient cell-free replication took place only with the homologous combinations.
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PMID:Cell-free replication of the human papillomavirus DNA with homologous viral E1 and E2 proteins and human cell extracts. 752 66

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
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PMID:Purification and properties of human DNA helicase VI. 754 99

Administration of hepatocarcinogens aflataxin B1 (AFB1) and N-nitrosodimethylamine (NDMA) to rats caused single-strand breaks in hepatic nuclear DNA. The damage was found to be maximum at 4 hours following AFB1 administration and at 2 hours following NDMA administration. These damages were repaired after 17 and 4 hours, respectively in cases of AFB1 and NDMA. The activity of poly(ADP-ribose)polymerase (PARP), an enzyme known to use single-strand breaks of DNA as cofactor, was observed to increase with increasing damage to DNA and decrease as and when this damage got repaired. DNA polymerase beta and DNA ligase activities were also seen to increase and decline in a way analogous to PARP. In contrast, DNA topoisomerase activity declined corresponding to an increase in PARP activity. These observations suggest a possible role of PARP in coordinating the activities of other enzymes involved in DNA repair. It is also envisaged that these parameters can be utilized to devise strategies to counteract the deleterious effects of chemical carcinogens.
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PMID:Activity of some nuclear enzymes associated with DNA repair following hepatocarcinogen administration to rats. 759 30

The mechanism for incorporation of aphidicolin-sensitive DNA polymerase into reconstituting sperm nuclei was studied in a Xenopus egg extract cell-free system. Aphidicolin-sensitive DNA polymerase activity was sedimented along with the light membrane fraction of Xenopus egg extract on a discontinuous sucrose gradient. Treatment of the egg extract with Triton X-100 caused DNA polymerase activity to migrate to a lighter density position at which free proteins were distributed. DNA polymerase activity was incorporated into the reconstituting sperm nuclei from the egg extract, but no nuclear incorporation was observed in nuclei incubated in egg extracts which had been treated with Triton X-100 or sonicated. The incorporation was also prohibited by several different treatments of the egg extract resulting in incomplete assembly of the nuclear membrane on the sperm nuclei. On the other hand, there was no inhibition of nuclear incorporation into the sperm nuclei reconstituting in the extracts which had been depleted of WGA-binding pore complex proteins or which contained a specific inhibitor of topoisomerase II (ICRF-193). In these two cases, the nuclear double-layered membrane assembled normally, although in the former case the sperm nuclei lacked lamina and did not initiate DNA replication, and in the latter case the sperm nuclei did not decondense but initiated DNA replication. Thus, it is concluded that DNA polymerase activity is incorporated into the reconstituting nuclei via the membraneous/particulate fraction of the egg extract simultaneously with nuclear double-layered membrane assembly. The lamina assembly and the transport system via the nuclear envelope pore complex are suggested not to participate in DNA polymerase nuclear incorporation.
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PMID:Aphidicolin-sensitive DNA polymerase is incorporated into the chromatin during nuclear envelope assembly in Xenopus egg extract. 762 44

Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.
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PMID:A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4. 768 30

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.
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PMID:Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins. 779 3

A new indolocarbazole antitumor agent, NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl) -5H- indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], enhanced the DNA cleavage catalyzed by HeLa S3 topoisomerase I at 0.01 microM but not the cleavage by topoisomerase II at 300 microM. It also caused single-strand DNA breakage in intact cells at 0.08 microM and more. Unlike the known topoisomerase I inhibitor camptothecin, NB-506 intercalated with DNA. However, the binding affinity to DNA and the inhibition against DNA polymerase alpha and RNA polymerase II were marginal compared with those of Adriamycin or actinomycin D. NB-506 inhibited the growth of various tumor cell lines at two micromoles or less, and its cytotoxicity was found to be cell line selective. This selective cytotoxicity of NB-506 was not fully explained by the differences in topoisomerase I activity in these cell lines, but there was some relationship between the amount of NB-506 accumulated in these cell lines and its cytotoxicity toward them. In conclusion, NB-506 is a potent topoisomerase I poison, acting selectively on tumor cell lines accumulating NB-506.
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PMID:Novel antitumor indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11- dihydroxy-13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4- c]carbazole-5,7(6H)-dione (NB-506): induction of topoisomerase I-mediated DNA cleavage and mechanisms of cell line-selective cytotoxicity. 788 28

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.
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PMID:DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4. 789 53

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.
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PMID:Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias. 791 48

We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.
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PMID:DNA replication from initiation zones of mammalian cells in a model system. 793 72


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