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)

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 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

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
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PMID:Biochemistry of homologous recombination in Escherichia coli. 796 21

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.
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PMID:Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan. 801 71

A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.
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PMID:Allosteric interaction of a herpes simplex viral thymidine kinase with host DNA polymerase alpha in mouse LP1-1 cells. 803 16

Aldehydes with specific protein-DNA crosslinking ability disrupted simian virus 40 (SV40) DNA replication to cause replication fork failure by the 40S intermediate pathway, in which replicating viral genomes become inactivated and torsionally stressed. In contrast, aldehydes without detectable protein-DNA crosslinking ability had no effect on SV40 DNA replication during the 10 min exposure times employed. This indicates that protein-DNA crosslinks block either DNA polymerase or the entire replication complex. Replication failure by the 40S pathway is known to initiate recombinational events in the damaged SV40 replicons. Similar events in cellular replicons may play a role in the clastogenic effects of formaldehyde. In addition, formaldehyde and acrolein caused accumulation of catenated (topologically linked) SV40 daughter chromosomes--a signature of topoisomerase II inhibition.
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PMID:Aldehyde-induced protein-DNA crosslinks disrupt specific stages of SV40 DNA replication. 820 64

Topoisomerase II (Top II) is the target enzyme for many antineoplastic drugs such as epipodophyllotoxins, anthracyclines, and acridines. Cell lines with alterations in Top II are resistant to drugs that interact with the enzyme. Studies of the Top II from a Chinese hamster ovary line, VpmR-5, that is resistant to VP-16 and VM-26, demonstrated that it is very similar, qualitatively and quantitatively, to its normal counterpart except that DNA cleavage by the VpmR-5 enzyme is not stimulated by VP-16 or VM-26. To understand the basis for the drug-resistant phenotype, the Top II cDNAs were isolated from both Chinese hamster ovary (CHO) and VpmR-5 cells by cDNA cloning with lambda gt22, and the entire cDNAs were sequenced. A mutation of G-->A at nucleotide 1478 was the only alteration observed in the VpmR-5 Top II cDNA compared with the wild-type gene. The mutation in VpmR-5 was confirmed by sequencing DNA fragments amplified from the genomic DNA by the polymerase chain reaction. Southern blot hybridization analysis of genomic DNA demonstrated loss of a Top II allele in VpmR-5 probably occurred during the development of resistance to etoposide. The mutation in VpmR-5 changes amino acid 493 from arginine to glutamine and is located adjacent to a putative ATP binding site of Top II. Mutations in an analogous region have been identified in two human leukemia cell lines by amplification of segments of Top II cDNA with Taq DNA polymerase. Taken together, these observations suggest that mutations in this region of the gyrase B domain of mammalian topoisomerase II may be capable of conferring resistance to antineoplastic agents that interact with this enzyme.
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PMID:Molecular cloning and identification of a point mutation in the topoisomerase II cDNA from an etoposide-resistant Chinese hamster ovary cell line. 838 May 92

An increase was observed in the total protein mass of nuclei isolated from Chinese hamster ovary cells heated at 45 degrees C or 45.5 degrees C. An increase in the fractional recovery of DNA polymerase alpha and beta, and of DNA topoisomerase activity coincided with this increase in the protein mass of nuclei from heated cells. Nuclear protein mass which was soluble in 2.0 M NaCl decreased 0.5 fold, while DNA-associated and nuclear matrix-associated protein mass increased 2.2 and 3.4 fold, respectively. The results indicate that the increase in nuclear protein mass observed in nuclei from heated cells is due in part to an increased binding, or precipitation, of nuclear proteins onto the cell's DNA and nuclear matrix.
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PMID:Nuclear protein redistribution in heat-shocked cells. 838 Nov 27


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