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)

Bleomycin (BLM) exclusively affects thymidine-containing compounds such as DNA and polydeoxyribonucleotides by releasing free thymine and leaving aldehyde functions. Molecular morphology and base sequence of the DNA strongly influence BLM activity. High BLM concentrations, besides modifying DNA into oligothyminic or athyminic nucleic acids, cause strand scissions. Enzymatic DNA and RNA synthesis is strongly influenced by BLM. The inhibition in DNA-dependent DNA polymerase and DNA-dependent RNA polymerase assays is of the non-competitive type. Protein biosynthesis in in vitro systems is not affected by BLM even at high concentrations. BLM turns out to be a strong inhibitor of DNase I and of DNase II; the inhibition is of the competitive type. The enzymatic activities of nucleases using RNA as substrate (RNase A, RNase B, Rnase T1, venom phosphodiesterase I and spleen phosphodiesterase II) are not influenced by this antibiotic. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro in low concentrations by cytostasis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells, DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo, using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This is attributed to the observed inhibition of DNA synthesis. RNA and protein synthesis as well as gene expression are not influenced by BLM under the conditions used. The selective inhibition of DNA synthesis in vivo may be caused by the following mechanisms: (1) competition of BLM with RNA; (2) blocking of the accessibility of DNA in chromatin to BLM, and (3) dependence from the repair processes. BLM inhibits growth of sarcomas, induced by oncogenic RNA viruses in vivo; well-developed tumours show regression after BLM treatment. Transformation of chick embryo fibroblasts by oncogenic RNA viruses in vitro and growth of these viruses is blocked by BLM; the most sensitive period for BLM inhibition is the time during the first period (integration of viral genome into cellular genome?) after infection.
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PMID:Effect of bleomycin on DNA, RNA, protein, chromatin and on cell transformation by oncogenic RNA viruses. 6 69

The ability of a series of haloalkanes, haloethanols and haloacetaldehydes to induce mutations in Salmonella typhrimurium and preferentially to inhibit the growth of DNA polymerase-deficient E. coli (pol A(+)/pol A(-)) was investigated. For the haloalkanes investigated, the order of reactivities towards the E. coli pol A(+)/pol A(-), was: 1,1,2,2-tetrabromoethane > 1,1-dibromoethane > 1,1,2,2-tetrachlorethane > 1,2-dibromoethane = 1,5 dibromopentane > 1,2-dibromo-2-methylpropane > 1-bromo-2-chloroethane > 1,2-dichloroethane. In the standard Salmonella mutagenicity assay the order of these substances was 1,2-dibromoethane = 1,5-dibromopentane > 1,2-dibromo-2-methylpropane >/= 1-bromo-2-chloroethane > 1,1,2,2-tetrachloroethane = 1,1-dibromoethane > 1,2-dichloroethane. 1,1,2,2-Tetrabromoethane was negative in the standard assay but strongly mutagenic when tested in suspension. It would appear that the discrepancy between the two procedures is due to the fact that bactericidal mutagens cannot be scored reliably in the standard Salmonella assay. The order of reactivity of 2-haloethanols in E. coli pol. A(+)/pol A(-), was 2-iodo > 2-bromo-> 2-chloroethanol. In the Salmonella assay the order was 2-bromo-> 2 iodo- >2-chloro-ethanol. 2-Fluoroethanol and ethanol were devoid of activity in both assays. For the 2-haloacetaldehydes the reactivities in the E. coli system were 2-bromoethylacetate > 2-bromoacetaldehyde = acetaldehyde > 2-chloroacetaldehyde while in the Salmonella system the order was 2-bromoethylacetate > 2-chloroacetaldehyde. Acetaldehyde had minimal activity, while 2-bromoacetaldehyde was without activity but strongly bactericidal.
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PMID:Mutagenicity of halogenated alkanes and their derivatives. 34 60

The mechanism by which the lipid peroxidation product 4-hydroxynonenal and several other homologous, yet non biogenic aldehydes inhibit proliferation of cultured Ehrlich ascites tumor cells has been studied. Incubation of cells (5 X 10(-4)/ml) in a minimum essential medium supplemented with 10 or 20 microM 4-hydroxynonenal reduces the 36-hr cell count to 65 and 30% of the control value. The reduced growth rate is most likely due to a blockage of the DNA synthesis. Cells labelled by a [3H]-thymidine pulse prior to exposure to 4-hydroxynonenal (20 microM, 8 hr) showed no change of the specific radioactivity of the DNA, indicating that no de novo synthesis occurred in the presence of the aldehyde. In the absence of the aldehyde the specific radioactivity of the DNA decreased by 25%. A 2-hr incubation in the presence of 10 or 20 microM of 4-hydroxynonenal reduced [3H]-thymidine incorporation into the HClO4 insoluble fraction to 85 and 50% of the controls, but had no effect of the [3H]-thymidine and 86Rb uptake. Moreover, examination of the cell cultures by the Trypan Blue exclusion technique revealed that 20 microM 4-hydroxynonenal does not cause cell death. The high reactivity of 4-hydroxynonenal towards sulfhydryl groups suggests that the aldehyde inhibits DNA synthesis by interacting with a functional SH group of DNA polymerase. The specific action on DNA synthesis is abolished at an aldehyde concentration of 50 microM, which leads to 30% (6 hr exposure) and 95% (36 hr exposure) of dead cells. The cytostatic index (CI), i.e. concentration at 50% Trypan Blue positive cells/concentration at 50% inhibition of cell growth deducted from the dose effect curves is 3.0 for 4-hydroxynonenal. The other homologous 4-hydroxyalkenals with chain length of 5, 6, 7, 8, 10 and 11 carbon atoms also inhibit cell growth. The CI varied from 1.20 to 1.94, indicating that these non biogenic 4-hydroxyalkenals have a distinctively lower specific effect on proliferation than the biogenic 4-hydroxynonenal. The Michael adducts of 4-hydroxynonenal with glutathione and cysteine were nearly one order of magnitude less toxic than the free aldehyde, the CI (2.41 cysteine adduct, 2.06 glutathione adduct), however, were not improved since the growth inhibitory action was also reduced.
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PMID:Effects of the lipidperoxidation product 4-hydroxynonenal and related aldehydes on proliferation and viability of cultured Ehrlich ascites tumor cells. 384 Jun 91

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.
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PMID:The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function. 618 18

We have shown that pyridoxal 5'-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.
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PMID:Divalent cation-dependent pyridoxal 5'-phosphate inhibition of Rauscher leukemia virus DNA polymerase: characterization and mechanism of action. 728 79

We have utilized acrolein as a model compound to examine the biochemical behavior of chemically-modified DNA polymerase alpha-primase complex (pol alpha). We have found that acrolein irreversibly inactivates the DNA synthetic capacity of pol alpha polymerase in a time- and concentration-dependent manner. Double-stranded DNA protects pol alpha polymerase from inactivation when present during acrolein exposure, but single-stranded DNA, dATP and ATP do not. Strikingly, the activity of pol alpha polymerase is strongly dependent upon the DNA substrate utilized to assay catalytic activity after exposure to the aldehyde. The primase activity of pol alpha is also inactivated by exposure to acrolein, but the observed rate of inactivation is slower than that seen for DNA synthesis. Competitive labeling studies with [14C] iodoacetamide suggest that acrolein inactivation of the enzyme is mediated through the modification of protein sulfhydryl groups.
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PMID:Inactivation of DNA polymerase alpha-primase by acrolein: loss of activity depends on the DNA substrate. 757 71

The purpose of this study was twofold: (1) to develop an optimized, reliable method for the flow cytometric analysis of the intranuclear DNA polymerase, terminal deoxynucleotidyl transferase (TdT) in acute myeloid leukemia, and (2) to establish the usefulness of a novel, fluorescein-isothiocyanate conjugated monoclonal anti-TdT antibody (HT-6) in double-fluorescence staining for surface antigens in the characterization of leukemic cells. Inclusion of an aldehyde blocking buffer in the staining protocol reduced background fluorescence sufficiently to allow for the detection of the low-level fluorescent TdT+ myeloblasts. When admixed to normal peripheral blood mononuclear cells, 0.4-0.5% of HLA-DR+ or myeloid surface antigen+, TdT+ double-stained myeloblasts could be reliably detected above background levels. Flow cytometric TdT measurements using the HT-6 antibody in 55 patients with TdT+ acute lymphocytic or myelocytic leukemia or blast crisis of chronic myelogenous leukemia were equal or superior to the results obtained with a mixture of monoclonal anti-TdT antibodies (anti-HTDT-Mix) and comparable to those obtained by the conventional slide method employing polyclonal rabbit anti-human TdT antiserum. This flow cytometric TdT determination in combination with surface antigen staining using a novel anti-TdT monoclonal antibody (HT-6) allows for the recognition of minimal leukemic blast cells during clinical remission in acute myeloid leukemia.
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PMID:Detection of terminal transferase in acute myeloid leukemia by flow cytometry. 792 95

Pyridoxal phosphate modification of adenovirus DNA polymerase results in loss of DNA polymerase activity, whereas the 3' --> 5' exonuclease activity is unaffected. Inhibition by pyridoxal phosphate is time-dependent, displays saturation kinetics, and is reversible in the presence of excess primary amine unless the pyridoxal phosphate-enzyme adduct is first reduced with NaBH4. Thus, inhibition is the consequence of Schiff base formation between the aldehyde moiety of pyridoxal phosphate and primary amino groups on the enzyme. In addition to inhibiting DNA polymerase activity, pyridoxal phosphate also inhibited the ability of the enzyme to initiate viral DNA replication, by transfer of dCMP onto the preterminal protein. Neither template-primer nor dNTP protect against pyridoxal phosphate inhibition, but the combination of template-primer and complementary substrate dNTP protected both initiation and DNA polymerase activities. Thus, it is likely that both the dCMP transfer activity required for initiation and DNA polymerase activity are carried out at the same site of the enzyme.
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PMID:Pyridoxal 5'-phosphate inhibition of adenovirus DNA polymerase. 879 69

The amino-terminal 8-kDa domain of vertebrate DNA polymerase beta (pol beta) has an activity to excise deoxyribose phosphate (dRP) groups from 5'-incised apurinic/apyrimidinic (AP) sites during base excision repair. The excision reaction proceeds via a beta-elimination reaction following formation of a Schiff base between an aldehyde group of the AP site and an amino group of the enzyme. Here we report that the Lys-72 residue of this enzyme is the catalytic center for dRP excision. Substitutions of Lys-72 with Arg or Gln reduced the dRP excision activity to less than 1% of the wild-type 8-kDa domain, while substitutions of Lys-35, Lys-68, or Lys-84 did not abolish its activity. The Lys-72 mutations also significantly decreased Schiff base intermediates trapped by reduction with sodium borohydride. The 8-kDa domain alone was able to bind preferentially to a single-nucleotide gap or 5'-incised synthetic AP site on double-stranded DNA. The Lys-72 mutations did not affect this damage-specific DNA binding activity. When introduced into the intact enzyme, a mutation of Lys-72 to Arg did not affect DNA synthesis activity of pol beta, but eliminated the repair activity. Addition of the wild-type 8-kDa domain to this reaction restored the repair activity. These results indicate a specific role of Lys-72 of pol beta in the dRP excision during base excision repair.
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PMID:Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups. 957 63

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.
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PMID:Excision of C-4'-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta. 978 84


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