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)

DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.
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PMID:Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity. 826 48

Protein kinase activity was revealed in complex forms of rat liver DNA polymerase alpha containing 3'-5'-exonuclease, primase, helicase, DNA ligase. Protein kinase (mol. mass about 200 kDa) has been partially purified from a specimen of high molecular mass DNA polymerase alpha of nuclear membrane of regenerating liver. The protein kinase activity of the complex form of DNA polymerase alpha was maximal in the cytosol in normal rat liver cells and in the nuclear membrane in dividing cells (40 h after partial hepatectomy). The main phosphokinase properties of this enzyme were determined.
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PMID:[Isolation of protein phosphokinase from a complex form of DNA polymerase alpha from rat liver]. 831 39

A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.
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PMID:A general two-metal-ion mechanism for catalytic RNA. 834 61

We have investigated the 3'-5'-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 5'-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.
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PMID:Digestion of damaged DNA by the T7 DNA polymerase-exonuclease. 834 24

Antibiotics, inhibitors of nucleic acids' synthesis from the group of chromomycins (olivomycin of sodium salt), anthracyclines (carminomycin and doxorubicin) and streptonigrin (bruneomycin) have been studied for their effect on DNA synthesis in vitro performed by DNA polymerases (1st and 2nd forms) of Acholeplasma laidlawii PG-8. It has been stated that olivomycin inhibits the function of both the first and second forms of DNA polymerases in proportion to an increase of the antibiotic concentration in the medium. Carminomycin in the concentration of about 1 microgram/ml almost completely inhibited the activity of both DNA polymerases. However, doxorubicin also belonging to the group of anthracyclins completely inhibited the activity of the first form of DNA polymerase in the concentration of 1 microgram/ml and practically has no effect in the concentration up to 100 micrograms/ml on the activity of the second form possessing 3'-->5'-function. Streptonigrin also proved to be suitable for differentiate the forms of DNA polymerases and to determine their functions. The first form of DNA polymerase with 5'-->3'-polymerase and exonuclease functions was not sensitive by this antibiotic in the concentration of 1000 micrograms/ml, while the activity of the second form of DNA polymerase with 3'-->5'-exonuclease functions was fully inhibited by this concentration of the antibiotic in the medium. The combination of doxorhubicin and streptonigrin in the medium can be used to determine the form of DNA polymerases and to identify their 5'-->3'- or 3'-->5'-exonuclease function and for selectivity inhibition of the function of one or another DNA polymerase in the medium.
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PMID:[Inhibitors of nucleic acid synthesis as a means of identifying the forms of DNA-dependent DNA polymerases in Acholeplasma laidlawii PG-8 and of determining their functions]. 835 26

A DNA polymerase with a 3'-to 5'-exonuclease that copurified with polymerase-primase from calf thymus was purified and extensively characterized. Its exonuclease degraded single-stranded DNA from 3' to 5' in a strictly distributive manner. On synthetic template-primer junctions, 3'-terminal mispairs were excised with a 10- to 20-fold preference over correctly paired nucleotides. In comparison to the 3'- to 5'-exonuclease the DNA polymerase activity was rather low. The ratio of nucleotides incorporated to nucleotides excised was in the order of 1 to 3 nucleotide insertions per excision, suggesting that net forward DNA synthesis is not the primary role of this DNA polymerase. DNA synthesis was performed with a low processivity in the presence and absence of PCNA. Both the polymerase and exonuclease activities were inhibited to a comparable extent by AMP. Thus, the exonuclease-polymerase might represent a novel DNA polymerase that we tentatively designate as DNA polymerase zeta. Possible benefits of DNA polymerase zeta in the process of error correction and the apparent dichotomy of an built-in proofreading activity for the processive DNA polymerases gamma, delta, and epsilon and an obviously external proofreading function for the less processive animal cell DNA polymerases alpha and beta are discussed.
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PMID:An error-correcting proofreading exonuclease-polymerase that copurifies with DNA-polymerase-alpha-primase. 838 85

The kinetic and functional characteristics of I and II forms of DNA-dependent DNA-polymerases of Acholeplasma laidlawii PG-8 have been studied. It is stated that I form of DNA polymerase possesses 5'-3'-exonuclease activity and is a typical replicase; II form of DNA-polymerase possesses both 5'-3'-polymerase and 3'-5'-exonuclease activity and is, evidently, a reparase. Both forms of enzyme give preference to poly(U)- and poly(A)-matrices having extremely high activity on these polymers. The enzymatic reactions realized by both forms of DNA-polymerases are described by the first-order equation. The calculated Michaelis-Menten constants equaled 180 and 250 microM for I and II forms of polymerases, respectively. It indicates that affinity to substrate in II form of polymerase is one-third higher than in I form of enzyme.
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PMID:[The kinetic and functional characteristics of DNA-dependent DNA-polymerases in Acholeplasma laidlawii PG-8]. 849 2

DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.
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PMID:DNA polymerase delta of Physarum polycephalum. 859 84

DNA polymerase epsilon (pol epsilon) was purified to apparent homogeneity from human placentas. The purified enzyme contains a single polypeptide of approximately 170 kDa (apparent mass) and has both DNA polymerase and 3'-5'-exonuclease activities. Competitive inhibition studies indicate that like DNA polymerases alpha and delta (pol alpha and pol delta, respectively), free pol epsilon binds single-stranded but not double-stranded DNA. This conclusion was confirmed by sedimentation binding analysis. Also like pol alpha and pol beta, pol epsilon exhibits induced dNTP inhibition in the presence of template annealed to complementary primer containing a 2',3'-H (dideoxy)-terminus. Together, these data suggest that pol epsilon follows an ordered sequential ter-reactant mechanism of substrate recognition and binding; it binds template first followed by annealed primer and then template-specified dNTP. Enzymologic studies suggest that in contrast to both pol alpha and pol delta, pol epsilon functions more efficiently as gap size decreases. This observation is consistent with a specific role for pol epsilon in gap-filling in vivo. Gap-filling is essential for both replication and repair.
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PMID:Human DNA polymerase epsilon: enzymologic mechanism and gap-filling synthesis. 863 8

Incorporation of the anticancer drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate; F-ara-AMP) into the 3'-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3' --> 5'-exonuclease activity of DNA polymerase epsilon from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase epsilon was unable to effectively remove F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase epsilon has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase epsilon, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase epsilon to the reaction. Furthermore, the addition of DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3' --> 5' exonuclease of human DNA polymerase epsilon can remove 3'-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."
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PMID:Inhibition of the 3' --> 5' exonuclease of human DNA polymerase epsilon by fludarabine-terminated DNA. 870 31


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