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)

The human DNA polymerase alpha catalytic polypeptide has been functionally overexpressed by a recombinant baculovirus in insect cells at greater than 1000-fold higher levels than that found in cultured normal human cells. The recombinant polymerase alpha protein is translated from its natural translation start codon under the control of the baculovirus polyhedron promoter producing a protein of 180 kDa, identical in size to that isolated from cultured human cells. This recombinant polymerase alpha is phosphorylated and reactive to a panel of monoclonal antibodies directed against the native polymerase alpha-primase complex and to polyclonal antisera against N- and C-terminal peptides of the polymerase alpha catalytic polypeptide. The recombinant enzyme was immunopurified from insect cells as a single polypeptide. The single subunit recombinant polymerase alpha has no detectable 3'-5' exonuclease activity. The Km for primer-template and dNTP, reactivity to inhibitors, N2-(p-n-butylphenyl)-dGTP (BuPdGTP) and aphidicolin, thermosensitivity, and DNA synthetic processivity and fidelity of the recombinant polymerase alpha are identical to that observed with the four-subunit polymerase alpha-primase complex immunopurified from cultured human cells. These results strongly suggest that the presence of the other subunits, (the p70 and the two primase subunits, p48 and p58), does not influence kinetic parameters of polymerase alpha catalysis, sensitivity to inhibitors, or DNA synthetic fidelity and processivity.
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PMID:Catalytic subunit of human DNA polymerase alpha overproduced from baculovirus-infected insect cells. Structural and enzymological characterization. 193 81

We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.
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PMID:The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. 198 82

The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.
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PMID:Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. 198 86

In addition to the general 3'-5' exonuclease domain described by Bernad et al. [Cell 59 (1989) 219-228] significant amino acid (aa) sequence similarity has been found in the C-terminal portion of 27 DNA-dependent DNA polymerases belonging to the two main superfamilies: (i) Escherichia coli DNA polymerase I (PolI)-like prokaryotic DNA polymerases, and (ii) DNA polymerase alpha-like prokaryotic and eukaryotic (viral and cellular) DNA polymerases. The six most conserved C-terminal regions, spanning approx. 340 aa, are located in the same linear arrangement and contain highly conserved motifs and critical residues involved in the polymerization function. According to the three-dimensional model of PolIk (Klenow fragment), these six conserved regions are located in the proposed polymerization domain, forming the metal and dNTP binding sites and the cleft for holding the DNA template. Site-directed mutagenesis in the phi 29 DNA polymerase supports some of these structural predictions. Therefore, it is likely that a 'Klenow-like core', containing the DNA polymerase and 3'-5' exonuclease activities, has evolved from a common ancestor, giving rise to the present-day prokaryotic and eukaryotic DNA polymerases.
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PMID:A general structure for DNA-dependent DNA polymerases. 205 76

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.
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PMID:The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase. 210 34

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.
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PMID:Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin. 212 28

In this report we have taken the reconstitution approach to study which subunits of the heterotrimer core polymerase (alpha, epsilon, theta) participate in the highly processive replication of long DNA templates by DNA polymerase III holoenzyme (holoenzyme). Comparison of the core and the alpha epsilon complex (the DNA polymerase and 3'-5' exonuclease subunits, respectively) shows they are both rapid and highly processive polymerases when they are reconstituted into a holoenzyme with the gamma complex (gamma delta delta' chi psi) and beta accessory proteins of holoenzyme. Specifically, holoenzyme reconstituted using either core or alpha epsilon completely replicates a uniquely primed 5.4-kilobase (kb) single-stranded DNA within 12 s in one binding event. Hence the theta subunit of core is not required for the processivity or speed of the holoenzyme. In contrast, when only the alpha subunit is reconstituted into the holoenzyme it is unable to replicate the entire 5.4-kb circle in one binding event but still retains a fairly high processivity of 1-3 kb and when given sufficient time for multiple binding events it finally finishes the entire circle. Therefore, highly processive DNA synthesis by holoenzyme is contingent on the epsilon exonuclease subunit. In light of these results the significance of the polymerase and exonuclease activities residing in two separate polypeptides is discussed.
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PMID:Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. 215 3

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.
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PMID:Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells. 215 92

DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism. Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity. Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated. When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated. This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing. A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair. Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit. A dGTPase activity was associated with the MutT protein. The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed.
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PMID:Molecular mechanisms of replicational fidelity in Escherichia coli. 215 94

The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5'-3'-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5'-3' direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5'-3'-exonucleolytic activities of the DNase and the polymerase-associated 3'-5' exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3'-5' exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.
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PMID:Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase. 216 60


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