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)

Water-soluble, octacationic zinc phthalocyanine (ZnPc) was found to be a very good G-quadruplex DNA stabilizer by using UV-melting studies and DNA polymerase stop assays, and a potent telomerase inhibitor by using the telomeric repeat amplification protocol (TRAP) assay. The compound's DNA-binding properties were also studied by surface plasmon resonance (SPR). Furthermore, CD experiments demonstrated that ZnPc could induce intramolecular G-quadruplex structure transition from the antiparallel to parallel form. More importantly, ZnPc was found to induce parallel structure formation in cation-deficient conditions. The stability of the induced structure was determined with CD melting assays.
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PMID:Quaternary ammonium zinc phthalocyanine: inhibiting telomerase by stabilizing G quadruplexes and inducing G-quadruplex structure transition and formation. 1736 82

DNA polymerases are enzymes responsible for the synthesis of DNA from nucleotides. Understanding their molecular fundamentals is a prerequisite for elucidating their aberrant activities in diseases such as cancer. Here we have carried out ab initio quantum mechanical/molecular mechanical (QM/MM) studies on the nucleotidyl-transfer reaction catalyzed by the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus, with template guanine and Watson-Crick paired dCTP as the nascent base pair. The results suggested a novel water-mediated and substrate-assisted (WMSA) mechanism: the initial proton transfer to the alpha-phosphate of the substrate via a bridging crystal water molecule is the rate-limiting step, the nucleotidyl-transfer step is associative with a metastable pentacovalent phosphorane intermediate, and the pyrophosphate leaving is facilitated by a highly coordinated proton relay mechanism through mediation of water which neutralizes the evolving negative charge. The conserved carboxylates, which retain their liganding to the two Mg2+ ions during the reaction process, are found to be essential in stabilizing transition states. This WMSA mechanism takes specific advantage of the unique structural features of this low-fidelity lesion-bypass Y-family polymerase, which has a more spacious and solvent-exposed active site than replicative and repair polymerases.
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PMID:A water-mediated and substrate-assisted catalytic mechanism for Sulfolobus solfataricus DNA polymerase IV. 1737 26

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg(332) in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg(332) with Ala, Glu, Leu, or His in order to probe the importance of Arg(332) in accurate and efficient bypass of 8-oxoG. The double mutant Ala(331)Ala(332) was also prepared to address the contribution of Arg(331). Transientstate kinetic results suggest that Glu(332) retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu(332) mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu(332) and the O-8 atom of 8-oxoG. The space normally occupied by Arg(332) side chain is empty in the crystal structures of the Ala(332) mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu(332) and Ala(332) mutants. These results support the view that a bond between Arg(332) and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.
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PMID:Hydrogen bonding of 7,8-dihydro-8-oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus solfataricus DNA polymerase Dpo4. 1746

Enzyme inhibition by fullerene derivatives has attracted much attention. In this communication, effects of two water-solube fullerene derivatives, fullerol and trimalonic acid C60 (TMA C60) on polymerase chain reaction (PCR) were investigated by using PCR of beta-actin cDNA derived from HeLa cells as an experimental model. Both fullerol and TMA C60 were found to inhibit PCR in a dose-dependent manner. PCR was ultimately inhibited while the concentrations of each compound were not less than 0.01 mM. In contrast, mannitol exerted no effects on PCR while its concentration increased up to 2 mM. Compensation experiments with Thermus aquaticus (Taq) DNA polymerase revealed that both fullerol and TMA C60 inhibited the enzymatic activity of Taq DNA polymerase, and the inhibitory potency of TMA C60 was slightly greater than that of fullerol. Our data provides some novel aspects on the enzyme inhibiting activities of fullerene derivatives.
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PMID:Inhibition of a thermophilic deoxyribonucleic acid polymerase by fullerene derivatives. 1767 10

DNA polymerases are crucial constituents of the complex cellular machinery for replicating and repairing DNA. Discerning mechanistic pathways of DNA polymerase on the atomic level is important for revealing the origin of fidelity discrimination. Mammalian DNA polymerase beta (pol beta), a small (39 kDa) member of the X-family, represents an excellent model system to investigate polymerase mechanisms. Here, we explore several feasible low-energy pathways of the nucleotide transfer reaction of pol beta for correct (according to Watson-Crick hydrogen bonding) G:C basepairing versus the incorrect G:G case within a consistent theoretical framework. We use mixed quantum mechanics/molecular mechanics (QM/MM) techniques in a constrained energy minimization protocol to effectively model not only the reactive core but also the influence of the rest of the enzymatic environment and explicit solvent on the reaction. The postulated pathways involve initial proton abstraction from the terminal DNA primer O3'H group, nucleophilic attack that extends the DNA primer chain, and elimination of pyrophosphate. In particular, we analyze several possible routes for the initial deprotonation step: (i) direct transfer to a phosphate oxygen O(Palpha) of the incoming nucleotide, (ii) direct transfer to an active site Asp group, and (iii) transfer to explicit water molecules. We find that the most probable initial step corresponds to step (iii), involving initial deprotonation to water, which is followed by proton migration to active site Asp residues, and finally to the leaving pyrophosphate group, with an activation energy of about 15 kcal/mol. We argue that initial deprotonation steps (i) and (ii) are less likely as they are at least 7 and 11 kcal/mol, respectively, higher in energy. Overall, the rate-determining step for both the correct and the incorrect nucleotide cases is the initial deprotonation in concert with nucleophilic attack at the phosphate center; however, the activation energy we obtain for the mismatched G:G case is 5 kcal/mol higher than that of the matched G:C complex, due to active site structural distortions. Taken together, our results support other reported mechanisms and help define a framework for interpreting nucleotide specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".
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PMID:DNA polymerase beta catalysis: are different mechanisms possible? 1769 33

Several quantum mechanical (QM) and hybrid quantum/molecular mechanical (QM/MM) studies have been employed recently to analyze the nucleotidyl transfer reaction in DNA polymerase beta (pol beta). Our examination reveals strong dependence of the reported mechanism on the initial molecular model. Thus, we explore here several model systems by QM methods to investigate pol beta's possible pathway variations. Although our most favorable pathway involves a direct proton transfer from O3'(primer) to O2alpha(Palpha), we also discuss other initial proton-transfer steps--to an adjacent water, to triphosphate, or to aspartic units--and the stabilizing effect of crystallographic water molecules in the active site. Our favored reaction route has an energetically undemanding initial step of less than 1.0 kcal/mol (at the B3LYP/6-31G(d,p) level), and involves a slight rearrangement in the geometry of the active site. This is followed by two major steps: (1) direct proton transfer from O3'(primer) to O2alpha(Palpha) leading to the formation of a pentavalent, trigonal bipyramidal Palpha center, via an associative mechanism, at a cost of about 28 kcal/mol, and (2) breakage of the triphosphate unit (exothermic process, approximately 22 kcal/mol) that results in the full transfer of the nucleotide to the DNA and the formation of pyrophosphate. These energy values are expected to be lower in the physical system when full protein effects are incorporated. We also discuss variations from this dominant pathway, and their impact on the overall repair process. Our calculated barrier for the chemical reaction clearly indicates that chemistry is rate-limiting overall for correct nucleotide insertion in pol beta, in accord with other studies. Protonation studies on relevant intermediates suggest that, although protonation at a single aspartic residue may occur, the addition of a second proton to the system significantly disturbs the active site. We conclude that the active site rearrangement step necessary to attain a reaction-competent geometry is essential and closely related to the "pre-chemistry" avenue described recently as a key step in the overall kinetic cycle of DNA polymerases. Thus, our work emphasizes the many possible ways for DNA polymerase beta's chemical reaction to occur, determined by the active site environment and initial models.
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PMID:A quantum mechanical investigation of possible mechanisms for the nucleotidyl transfer reaction catalyzed by DNA polymerase beta. 1776 65

Phospholipid fatty acid (PLFA) analysis and 16S ribosomal DNA polymerase chain reaction amplification-denaturing gradient gel electrophoresis (PCR-DGGE) were used to determine microbial communities and predominant microbial populations in water samples collected from a pilot-scale constructed wetland system. This pilot-scale constructed wetland system consists of three types: subsurface-flow (SSF), surface-flow (SF) and a floating aquatic plant (FAP) system. Analysis of PLFA profiles indicated primarily eukaryotic organisms, including fungi, protozoa, and diatoms were observed in all three wetland systems. Biomarkers for Gram-negative bacteria were also detected in all samples analyzed while low proportions of biomarkers for Gram-positive bacteria were observed. Biomass content (total PFLA/sample) was highest in water samples collected from both SF and FAP system while highest metabolic activity was observed in FAP system. This is consistent with the observed highest metal removal rate in FAP system. Sequence analysis of the predominant PCR-DGGE DNA fragments showed 0.92 to 0.99 similarity indices to Beta-proteobacteria, Flavobacterium sp. GOBB3-206, Flexibacter-Cytophaga-Bacteroides group, and Gram-positive bacteria. Results suggest diverse microbial communities including microorganisms that may significantly contribute to biogeochemical elemental cycles.
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PMID:Characterization of microbial communities in a pilot-scale constructed wetland using PLFA and PCR-DGGE analyses. 1784 6

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.
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PMID:Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization. 1878 38

The selective detection of the anion pyrophosphate (PPi) is a major research focus. PPi is a biologically important target because it is the product of ATP hydrolysis under cellular conditions, and because it is involved in DNA replication catalyzed by DNA polymerase, its detection is being investigated as a real-time DNA sequencing method. In addition, within the past decade, the ability to detect PPi has become important in cancer research. In general, the sensing of anions in aqueous solution requires a strong affinity for anions in water as well as the ability to convert anion recognition into a fluorescent or colorimetric signal. Among the variety of methods for detecting PPi, fluorescent chemosensors and colorimetric sensors for PPi have attracted considerable attention during the past 10 years. Compared with the recognition of metal ions, it is much more challenging to selectively recognize anions in an aqueous system due to the strong hydration effects of anions. Consequently, the design of PPi sensors requires the following: an understanding of the molecular recognition between PPi and the binding sites, the desired solubility in aqueous solutions, the communicating and signaling mechanism, and most importantly, selectivity for PPi over other anions such as AMP and ADP, and particularly phosphate and ATP. This Account classifies chemosensors for PPi according to topological and structural characteristics. Types of chemosensors investigated and reported in this study include those that contain metal ion complexes, metal complexes combined with excimers, those that function with a displacement approach, and those based on hydrogen-bonding interaction. Thus far, the utilization of a metal ion complex as a binding site for PPi has been the most successful strategy. The strong binding affinity between metal ions and PPi allows the detection of PPi in a 100% aqueous solution. We have demonstrated that carefully designed receptors can distinguish between PPi and ATP based on their different total anionic charge densities. We have also demonstrated that a PPi metal ion complex sensor has a bioanalytical application. This sensor can be used in a simple and quick, one-step, homogeneous phase detection method in order to confirm DNA amplification after polymerase chain reaction (PCR).
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PMID:Chemosensors for pyrophosphate. 1879 56

A new label-free, homogenous, sensitive and economical one-step method to detect SNP genotyping of genomic DNA has been developed by combining allele-specific PCR technique with water-soluble cationic conjugated polyelectrolytes (CCP). The amplification of target DNA and fluorescence detection steps are combined into one-step. The target DNA fragment containing a G allele site acts as PCR template. For the G allele-specific forward primer whose 3'-terminal base is complementary to the G allele template, after the first step of reverse primer extension, G allele-specific primer perfectly anneals with newly formed strand and the extension reaction of forward primer starts. During the extension, dGTP-Fl and dUTP-Fl are incorporated into extension chain in the presence of Taq DNA polymerase and more fluorescein-labeled PCR amplicons are yielded. Upon adding the CCP, strong electrostatic interactions between DNA and CCP bring them close and efficient FRET from CCP to fluorescein occurs. For the C allele-specific forward primer, less fluorescein-labeled PCR amplicons are yielded and inefficient FRET occurs. By triggering the change of emission intensity of CCP and fluorescein, it is possible to assay the SNP genotypes. In contrast to previous reports, this method does not require designing dye-labeled primers, and gel electrophoresis and isolation step after PCR were avoided in this homogenous method. The genotyping of 50ng genomic DNA from human lung cancer cell is easily detected using our new method. These qualities will make the new detection system ideal for SNP genotyping.
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PMID:Homogeneous and one-step fluorescent allele-specific PCR for SNP genotyping assays using conjugated polyelectrolytes. 1907 Apr 77


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