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)

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.
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PMID:Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision. 166 14

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.
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PMID:NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment. 226 37

A new method of estimation of dissociation constants for ligands and free energies of its binding based on the affinity modification of active centers in the presence of competitive ligands was developed. This method is designed for the analysis of protein-nucleic acid interactions in template systems. Deoxyoligoribonucleotides containing the reactive residue of cis-aquadihydroxydiaminoplatinum (II) and oligonucleotides ethylated at phosphate groups were used for the study of interactions of human placental DNA-polymerase alpha and the Klenow fragment of DNA-polymerase I from E. coli with templates and primers. A model was constructed which postulates the formation of a single Me2+-dependent electrostatic bond and of a hydrogen bond by one of template phosphates with the enzyme active center. Similar bonds form the basis for the enzyme interaction with the 3'-terminal phosphate group of the primer. Other monomeric units of the template are likely to interact with the enzyme by forming hydrophobic bonds. Other mononucleotide units of the primer are involved in complementary interactions with the template. The primer activity of dNMP and NMP in these systems has been demonstrated for the first time. The efficiency of dNMP, dNDP and dNTP interaction with DNA-polymerase was estimated from the affinity modification of the enzymes by dNTP and dNMP imidazolides. The key role of the template-primer interaction in the formation of the dNTP-binding site of DNA-polymerases was demonstrated. A significant contribution of dNTP gamma-phosphate to the template--dependent specific tuning of substrate dNTP was revealed.
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PMID:[Protein-nucleic acid interactions in reactions catalyzed by eukaryotic and prokaryotic DNA-polymerases]. 250 66

One of the two forms of DNA polymerase alpha from ovaries of the frog Xenopus laevis catalyzed ribonucleoside triphosphate-dependent DNA synthesis on single-stranded circular fd phage DNA templates. DNA synthesis was dependent on ATP and added template. CTP, GTP, and UTP stimulated DNA synthesis but were not required and could not substitute for ATP. DNA synthesis was not inhibited by alpha-amanitin. Neither poly(dT) nor double-stranded DNA served as template. Analysis of [32P]-dTMP-labeled product by neutral and alkaline agarose gel electrophoresis showed that 0.1- to 1-kilobase DNA fragments (average size of approximately equal to 0.25 kilobase) were synthesized. The fragments were not covalently linked to the template. Either [alpha-32P]NMP, [gamma-32P]ATP, or [gamma-32P]GTP were incorporated also into the product. Analysis of the product after hydrolysis by KOH, alkaline phosphatase, or bacteriophage T4 3' leads to 5' exonuclease showed the presence of a small oligoribonucleotide primer at the 5' end of the newly synthesized DNA. NTP-dependent DNA-synthesizing activity copurified on six columns and cosedimented during glycerol gradient centrifugation with one form of DNA polymerase alpha activity but not with the other form. These results suggest that DNA primase activity is associated with one of the two forms of X. laevis DNA polymerase alpha.
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PMID:DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries. 696 3

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.
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PMID:Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. 812 24

Anaplastic large cell lymphoma (ALCL) is a distinct clinicopathologic variant of intermediate grade non-Hodgkin's lymphomas (NHL) composed of large pleomorphic cells that usually express the CD30 antigen and interleukin (IL)-2 receptors, and is characterized by frequent cutaneous and extranodal involvement. With variable frequency ALCL bear the t(2;5)(p23;q35) chromosomal translocation that fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a novel protein kinase gene, Anaplastic Lymphoma Kinase (ALK), on chromosome 2p23. We determined the frequency of this translocation with a novel DNA polymerase chain reaction (PCR) technique using 0.5 microgram of genomic DNA, 5'-primers derived from the NPM gene and 3'-primers derived from the ALK gene and hybridization with internal probes. The presence of amplifiable DNA in the samples was tested with the inclusion in the PCR reaction of oligonucleotide primers designed to amplify a 3016-bp fragment from the beta-globin locus. NMP-ALK fusion amplicons were detected using DNA isolated either from all three ALCL cell lines tested, or from all four primary ALCL tumors known to contain the t(2;5)(p23;q35) translocation. Nested amplicons were detected by hybridization in 100% of specimens diluted 10(4)-fold and in 20% of those diluted 10(5)-fold. We subsequently examined archival genomic DNA from 20 patients with ALCL, 39 with diffuse large cell, 2 with mantle cell, 20 with peripheral T cell, 13 with low-grade NHL, 31 with Hodgkin's disease (HD), and 6 with lymphomatoid papulosis. Fusion of the NPM and ALK genes was detected in three of 18 patients with ALCL who had amplifiable DNA (17%, 95% confidence intervals 4% to 41%), but not in any patients with other NHL, HD, or lymphomatoid papulosis. The amplicon sizes were different in all cell lines and patients reflecting unique genomic DNA breakpoints. We conclude that with genomic DNA-PCR the rearrangement of the NPM and ALK loci is restricted to patients with ALCL. Further studies are needed to determine the prognostic significance of the NPM-ALK rearrangement, to determine whether its detection can aid in the differential diagnosis between ALCL. Hodgkin's disease, and lymphomatoid papulosis, and to establish the usefulness of the genomic DNA PCR in the monitoring of minimal residual disease in those patients whose tumors bear the t(2;5).
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PMID:Amplification of genomic DNA demonstrates the presence of the t(2;5) (p23;q35) in anaplastic large cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis. 926 95

Substrate specificities of three viral replicative polymerases of different origins (HIV reverse transcriptase, hepatitis C virus RNA polymerase, and herpes virus DNA polymerase) towards 2'F-NTP were studied. Activated DNA, polyA-oligoUs and (2'F-A)20-oligoU6-complexes were used as templates. It was shown that all DNA polymerases studied can incorporate 2'F-NMP into the 3'-end of primer-template complexes. HIV reverse transcriptase and herpes virus DNA polymerase can elongate synthesis with both dNTP and 2'F-NTP. Homopolymer (2'F-A)20 can serve as a template for polymerization of both UTP and 2'F-UTP,-catalyzed by hepatitis C virus polymerase although with efficacy about 5 to 10-fold lower in comparison with natural primertemplate complex. Pyrophosphorolysis reaction of 2'F-CMP residue at 3'-end of primer catalyzed with HIV reverse transcriptase is going by two orders of magnitude less effective if compared with natural dNMP residue at the same system.
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PMID:[2'fluoro derivatives of nucleosides as substrates of viral replicative nucleotide polymerases]. 2584 69