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 polymerases play a central role in the mechanisms of DNA replication and repair. Here, we report mechanisms of the beta-polymerase catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA. Based on energy minimizations, molecular dynamics simulations, and free energy calculations of solvated ternary complexes of pol beta and by employing a mixed quantum mechanics molecular mechanics Hamiltonian, we have uncovered the identities of transient intermediates in the phosphoryl transfer pathways. Our study has revealed that an intriguing Grotthuss hopping mechanism of proton transfer involving water and three conserved aspartate residues in pol beta's active site mediates the phosphoryl transfer in the correct as well as misincorporation of nucleotides. The significance of this catalytic step in serving as a kinetic check point of polymerase fidelity may be unique to DNA polymerase beta, and is discussed in relation to other known mechanisms of DNA polymerases.
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PMID:Mechanism of nucleotide incorporation in DNA polymerase beta. 1706 55

The prokaryotic DNA polymerase III clamp loader complex loads the beta clamp onto DNA to link the replication complex to DNA during processive synthesis and unloads it again once synthesis is complete. This minimal complex consists of one delta, one delta' and three gamma subunits, all of which possess an AAA+ module--though only the gamma subunit exhibits ATPase activity. Here clues to underlying clamp loader mechanisms are obtained through Bayesian inference of various categories of selective constraints imposed on the gamma and delta' subunits. It is proposed that a conserved histidine is ionized via electron transfer involving structurally adjacent residues within the sensor 1 region of gamma's AAA+ module. The resultant positive charge on this histidine inhibits ATPase activity by drawing the negatively charged catalytic base away from the active site. It is also proposed that this arrangement is disrupted upon interaction of DNA with basic residues in gamma implicated previously in DNA binding, regarding which a lysine that is near the sensor 1 region and that is highly conserved both in bacterial and in eukaryotic clamp loader ATPases appears to play a critical role. gamma ATPases also appear to utilize a trans-acting threonine that is donated by helix 6 of an adjacent gamma or delta' subunit and that assists in the activation of a water molecule for nucleophilic attack on the gamma phosphorous atom of ATP. As eukaryotic and archaeal clamp loaders lack most of these key residues, it appears that eubacteria utilize a fundamentally different mechanism for clamp loader activation than do these other organisms.
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PMID:Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine. 1701 86

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.
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PMID:Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer. 1713 89

The fresh water crustacean Daphnia magna is widely used as a test organism in aquatic toxicology to assess the adverse effects of individual substances or complex mixtures, e.g. industrial wastewaters. Cultures are held in several European testing laboratories and testing is typically carried out according to internationally standardised protocols. However, despite accounting for many potential confounding factors these guidelines do not currently take into account any specification related to the use of a specific clone. Cultures from seven laboratories were used to assess genetic variability by random-amplified polymorphic DNA polymerase chain reaction. Results pointed out the existence of two main clone clusters Responses in the acute Daphnia immobilisation test showed no direct correlation with genetic clusters resulting from random genetic markers (random-amplified polymorphic DNA) analysis. Considering that genetic differences are the most probable cause for the ecotoxicological test data, further analysis concerning gene expression and genetic stability should be performed.
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PMID:Genetic variability in Daphnia magna and ecotoxicological evaluation. 1713 28

With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.
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PMID:Regulation of DNA repair fidelity by molecular checkpoints: "gates" in DNA polymerase beta's substrate selection. 1717 36

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.
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PMID:UTP-bound and Apo structures of a minimal RNA uridylyltransferase. 1718 40

The mechanism of the fidelity synthesis of DNA associated with the process of dGTP combination to the DNA template was explored. The exclusion of water molecules from the hydrated DNA bases can amplify the energy difference between the correct and incorrect base pairs, but the effect of the water molecules on the Gibbs free energy of formation is dependent on the binding sites for the water molecules. The water detachment from the incoming dNTP is not the only factor but the first step for the successful replication of DNA. The second step is the selection of the DNA polymerase on the DNA base pair through the comparison between the correct DNA base and the incorrect DNA base. The bonding of the Arg668 with the incoming dNTP can enlarge the Gibbs free energies of formation of the base pairs, especially the correct base pairs, thus increasing the driving force of DNA formation. When the DNA base of the primer terminus is correct, the extension of the guanine and the adenine is quicker than that of the cytosine and the thymine because of the hydrogen bonding fork formation of Arg668 with the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP. Because of the geometry differences of the incorrect base pairs with the correct base pairs, the effect from the DNA polymerase is smaller on the incorrect base pair than on the correct base pair, and the extension of a mispair is slower than that of a correct base pair. This decreases the extension rate of the base pair and thus allows proofreading exonuclease activity to excise the incorrect base pair. Arg668 cannot prevent the extension of the GT mispair, as well as the GC correct base pair, and GA and GG mispairs. This may be attributed to the small geometry difference between the GT base pair and the correct AT base pair.
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PMID:Factors determining the deriving force of DNA formation: geometrical differences of base pairs, dehydration of bases, and the arginine assisting. 1726 49

In the family-B DNA polymerase of bacteriophage RB69, the conserved aromatic palm-subdomain residues Tyr391 and Tyr619 interact with the last primer-template base-pair. Tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. The main-chain amide of Tyr391 interacts with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. We investigated the roles of these residues in replication fidelity in vivo employing phage T4 rII reversion assays and an rI forward assay. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so we used an in vitro fidelity assay. Tyr391F/M replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on single-stranded DNA and double-stranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3' primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis.
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PMID:The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity. 1732 43

Reliable and efficient PCR and extension reactions using standardized procedures are key elements for successful single nucleotide polymorphism (SNP) genotyping projects. To improve the cost efficiency and overall performance of SNP genotyping we evaluated two commercial thermostable DNA polymerases used for the extension reaction in the homogeneous mass extension MassARRAY genotyping system. The aim was to study whether the quality, accuracy, and expenses of a new TERMIPol DNA polymerase are competitive to the commonly used ThermoSequenase DNA polymerase. We compared the enzymes by testing 96 SNPs genotyped for DNA samples of 31 unrelated individuals and one water control. The success rates, congruence between the genotypes and completeness of extension reactions support the use of TERMIPol, especially when the amplification of the higher mass allele is difficult. Further, using TERMIPol enabled successful genotyping (>93%) of several SNPs that failed (<80% success) when using ThermoSequenase.
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PMID:The enhancement of homogenous mass extension reaction: comparison of two enzymes. 1733 99

The rate-limiting step for nucleotide incorporation in the pre-steady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D(pol)), chemistry is partially (Mg(2+)) or completely (Mn(2+)) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK(a) values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3'-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3'-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.
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PMID:Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. 1736 May 13


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