Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the mechanism by which the 8-hydroxyguanine residue in DNA affects the fidelity of DNA replication, the intrinsic properties of this modified base were investigated using an ab initio molecular orbital method. The most stable 8-hydroxyguanine form was revealed to be 6,8-diketo. The addition of an oxygen atom to the 8 position of a guanine base was shown to change the electrostatic potential of the molecule entirely and to give it a negative character. This effect may influence the local structure of 8-hydroxyguanine-containing DNA and the interaction with DNA polymerase, thereby resulting in infidelity of DNA replication.
 ...
PMID:An ab initio molecular orbital study on the characteristics of 8-hydroxyguanine. 365 46

A variety of factors were found to modify the toxicity of L-dopa in HeLa cells (D37 16 microM) and in dopa-sensitive, nonpigmented human melanoma cells (MM96) (D37 5 microM) having a similar size and doubling time. Dopa toxicity was decreased by concurrent treatment with superoxide dismutase, peroxidase or catalase, by erythrocytes, or by hypoxia. Toxicity could be increased by the enzyme inhibitors L- and D-penicillamine, sodium diethyldithiocarbamate or 3-amino-1,2,4-triazole. The two cell lines had similar levels of superoxide dismutase and peroxidase; in 6 human melanoma lines, no correlation was found between dopa killing and tyrosinase activity as determined either by formation of dopa from tyrosine or by formation of melanin from dopa. Uptake of L-dopa was similar in HeLa and MM96 cells, and the toxicity of D-dopa was the same in both lines as that of the L-isomer. Dopa decomposed within 12 hr in culture medium, the rate and products being influenced by addition of the above enzymes and by the cell density. Dopa-melanin and medium containing decomposed dopa were also selectively toxic to MM96 cells. Adenovirus 5 was used in two different ways to assess the relative importance of DNA damage and inhibition of DNA synthesis by dopa. Viral replication was found to be unaffected in cells being treated with dopa but was strongly inhibited in cells treated with the DNA polymerase inhibitor cytosine arabinoside. Secondly, the virus was itself inactivated by treatment with dopa for 24 hr (D37 1.3 mM); similar dose response curves were obtained for replication of dopa-treated virus in untreated HeLa or MM96 cells. These results show that the initial events of dopa toxicity occur outside the cell and lead to the formation of a stable, toxic product (probably melanin) which does not strongly inhibit DNA polymerase activity. Melanoma hypersensitivity was not due to differences in oxygen-metabolizing enzymes, dopa uptake, or DNA repair.
 ...
PMID:Modification of dopa toxicity in human tumour cells. 392 49

The time scale for rejoining of radiation-induced deoxyribonucleic acid (DNA) single-strand breaks was measured in the presence and absence of oxygen. The involvement of DNA polymerase I in this repair process was studied. Formation and rejoining of DNA strand breaks were measured in lambda DNA infecting lysogenic pol(+) and polA1 strains of Escherichia coli irradiated by 4 MeV electrons under identical conditions. Irradiation and transfer to alkaline detergent could be completed in less than 180 ms. The initial yields of DNA strand breaks were identical in pol(+) and polA1 host cells and four- to fivefold higher in the presence of oxygen than in nitrogen anoxia. Evidence for the existence of a very fast repair process, independent of DNA polymerase I, was not found, since no rejoining of radiation-induced DNA strand breaks was observed during incubation from 45 ms to 3 s. In pol(+) host cells most of the strand breaks produced in the presence of oxygen were rejoined within the first 30 to 40 s of incubation, whereas no rejoining could be detected within the same period of time in anoxic cells. Since no rejoining of broken lambda DNA molecules was observed in polA1 host cells, it is concluded that the synthetase activity of DNA polymerase I is involved in the rejoining of DNA breaks induced by radiation in the presence of oxygen.
 ...
PMID:Time scale for rejoining of bacteriophage lambda deoxyribonucleic acid molecules in superinfected pol+ and polA1 strains of Escherichia coli after exposure to 4 MeV electrons. 460 87

(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.
 ...
PMID:Stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. 609 2

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.
 ...
PMID:Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA. 623 Oct 54

The contribution of proofreading to the fidelity of catalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thio]triphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the alpha phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with phi X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase beta (enzymes without a proofreading 3' leads to 5' exonucleolytic activity), substitution of deoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokaryotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading of a C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.
 ...
PMID:Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis. 645 18

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.
 ...
PMID:Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. 753 90

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
 ...
PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

We found that hydroxylation occurs at the C-2 position of adenine by oxygen radical treatment (Fe2+-EDTA) of dA, dATP, and single- and double-stranded DNA. This oxidatively damaged base, 2-hydroxyadenine, was produced 3-6-fold and 40-fold less than 8-hydroxyguanine when monomers and polynucleotides, respectively, were treated. To determine whether the damaged nucleotide, 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into a growing DNA, and to reveal the kinds of nucleotides opposite which 2-OH-dATP is incorporated, calf thymus DNA polymerase alpha and the Klenow fragment of Escherichia coli DNA polymerase I were used in vitro DAN synthesis in the presence of 2-OH-dATP. DNA polymerase alpha incorporated the nucleotide opposite T and C in the DNA template. On the other hand, in an experiment using the Klenow fragment, incorporation of 2-OH-dATP was observed only opposite T. Steady-state kinetic studies indicated that incorporation of 2-OH-dATP by DNA polymerase alpha opposite T was favored over that opposite C by a factor of only 4.5. These results indicate that 2-OH-dATP, an oxidatively damaged nucleotide, is a substrate for DNA polymerases and is incorporated incorrectly by the replicative DNA polymerase.
 ...
PMID:Formation of 2-hydroxydeoxyadenosine triphosphate, an oxidatively damaged nucleotide, and its incorporation by DNA polymerases. Steady-state kinetics of the incorporation. 764 27

We hypothesized that cellular proliferation and the capacity to repair DNA damage in the lung might differ during the pre- and postnatal periods, because the lung is exposed to higher oxygen concentrations and/or various mutagens after birth. In order to test this hypothesis, changes in DNA content and the activities of DNA polymerase alpha and beta were studied in the lungs of 1-day prenatal to 42-day postnatal rats. Total DNA polymerase activity reached its highest level at 1 day prenatal and 1 day after birth. The activity decreased exponentially by 28% up to 14 days of age, a change inversely related to the change in DNA content. The change in total DNA polymerase activity agreed closely with the change in DNA polymerase alpha activity, but not the activity of the beta form, although small elevations in both DNA polymerase alpha and beta were observed on day 3, possibly reflecting the mechanical effect of delivery. The activity of DNA polymerase beta remained relatively constant from 1 day before birth to 21 days after birth, varying by only about 5%. From these results, it is concluded that: (1) cellular proliferation in the lung is most active during the first 2 weeks after birth as supported by the increases in DNA polymerase alpha activity and DNA content, and (2) anticipating the oxygen enriched atmosphere after birth, the level of DNA polymerase beta, involved in the DNA repair system, is already elevated during the prenatal period and remains constant throughout the postnatal period.
 ...
PMID:Changes in the activities of DNA polymerases in growing rat lungs. 780 74


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>