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)

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.
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PMID:Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein. 831 57

Peptide I, a 50-amino acid synthetic peptide based on residues 728 to 777 of DNA polymerase I, binds dNTP substrates and duplex DNA (G. Mullen, P. Shenbagamurthi, and A.S. Mildvan, J. Biol. Chem. 264, 19637-19647, 1988). The structural properties of peptide I at pH 3.9 have been studied by CD spectroscopy and by 2D proton NMR at 600 MHz. The CD spectra are fit by assuming that peptide I contains 17% helix, 17% beta-structure, and 66% coil. The substrate dATP binds tightly to peptide I under these conditions (KD = 0.5 microM) as determined by fluorescence quenching but induces no change in peptide conformation, as detected by CD spectroscopy. Proton resonances of peptide I have been assigned by double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy. As found with other peptides, peptide I is best characterized by both extended and partially folded secondary structures which equilibrate rapidly on the NMR time scale. A region from residues 3 through 10 displays nuclear Overhauser effects (NOEs) consistent with the rapid equilibration of a nascent helix with a random extended structure. Alternatively this segment of residues is consistent with a series of three opened-out turns. A nonclassical turn is found between residues 14 and 17 and from residues 44 to 47, the latter closing irregular antiparallel strands from residues 42 to 48. The remainder of the peptide is a coil. A residue-by-residue comparison of the best-fit solution structure of the peptide with that of the corresponding sequence in the X-ray structure of the complete enzyme reveals that 36% of the amino acids are found to be in a conformation similar to that in the enzyme. Such partial and transient folding of the peptide indicates that the major role of the remainder of the protein is to provide structural support for the active site region of the enzyme. As detected by interresidue NOEs and NOEs to water protons, the homologous sequence Leu-37-Ile-38-Tyr-39-Gly-40, together with Phe-15 of the peptide, provides an exposed hydrophobic cluster of residues which may constitute the substrate binding site. An exposed cluster of cationic residues consisting of Arg-27, Arg-28, Lys-31, and possibly Arg-48 may provide the binding site for duplex DNA.
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PMID:Sequential proton NMR resonance assignments, circular dichroism, and structural properties of a 50-residue substrate-binding peptide from DNA polymerase I. 844 59

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.
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PMID:Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function. 862 6

DNA polymerase beta (beta-Pol) consists of an N-terminal ssDNA binding domain with deoxyribose phosphodiesterase activity and a C-terminal domain with nucleotidyltransferase activity. The solution structure of the cloned N-terminal domain of beta-Pol has been determined by multidimensional heteronuclear NMR using experimental restraints that included 1030 distances based on analysis of NOE connectivities, 68 phi, chi 1, and chi 2 torsion angles based on analysis of couplings, and 22 hydrogen bonds. Hydrogen bonds were assessed only within helices by the absence of saturation transfer from water at pH 6.7, by NOEs and JNH alpha couplings indicative of well-structured helices, and by 13C alpha chemical shifts characteristic of helices. The root mean square deviation for heavy backbone atoms within the helices was 0.64 A in 55 structures. The solution structure of the N-terminal domain is formed from four helices packed as two antiparallel pairs crossing at 50 degrees in a V-like shape. The domain binds p(dT)8, a template analogue, as a 1:1 complex in 100 mM NaCl (KD = 10 microM). Analysis of the binding equilibria at increasing NaCl concentrations indicated that ionic contacts contribute to the complex. The binding interaction was mapped to one face of the domain by characterizing backbone 1H and 15N chemical shift changes. Assigned intermolecular NOEs from 2D NOESY support the assessment of the binding interface. The structure that forms the interaction surface includes an antiparallel helix-3-turn-helix-4 motif and residues adjacent to an omega-type loop connecting helix-1 and helix-2. Sites appropriate for nucleotide contact on the structure are described. The mapped interaction interface for a ssDNA template is the first described for a DNA polymerase.
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PMID:Three-dimensional solution structure of the N-terminal domain of DNA polymerase beta and mapping of the ssDNA interaction interface. 863 59

DNA polymerase makes errors by misincorporating natural DNA bases and base analogs. Because of the wide variety of possible mismatches and the varying efficiency with which they are repaired, structural studies are necessary to understand in detail how these mispairs differ and can be distinguished from standard Watson-Crick base pairs. 2-Aminopurine (AP) is a highly mutagenic base analog. The objective of this study was to determine the geometry of the AP x C mispair in DNA at neutral pH. Although several studies have focused on the AP x C mispair in DNA, there is not as of yet consensus on its structure. At least four models have been proposed for this mispair. Through the use of NMR spectroscopy with selective 15N-labeling of exocyclic amino nitrogens on bases of interest, we are able to resolve ambiguities in previous studies. We find here that, in two different DNA sequences, the AP x C mispair at neutral and high pH is in a wobble geometry. The structure and stability of this base mispair is dependent upon the local base sequence.
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PMID:NMR study of the conformation of the 2-aminopurine:cytosine mismatch in DNA. 867 36

A high-resolution structure of a 16-nucleotide bacteriophage T4 RNA hairpin, 5'-GCCU[AAUAACUC]GGGC (loop bases in square brackets), has been determined in solution by proton, phosphorus, and carbon (natural abundance) NMR spectroscopy. This RNA hairpin is known to play a crucial role in the translational repression of bacteriophage T4 DNA polymerase. Ultraviolet absorbance melting curves indicate that the structure formed is unimolecular. The NMR spectra indicate that a single conformation consistent with a hairpin structure is formed. Strong imino-imino NOEs confirm the formation of the G.U base pair at the stem-loop junction. There is no evidence that A5 is protonated (at pH 6.0) and involved in an A+.C pair. However, the NMR data indicate that the stem is extended beyond the G.U pair and that A-form stacking continues for three nucleotides on the 5' side and one nucleotide on the 3' side. Structure calculations using restraints obtained from NMR data give a precisely defined structure with an average root mean square deviation (RMSD) of approximately 1.2 A for the entire molecule. The assignment of all the protons and most of the 31P resonances in the loop yielded a large number of distance and torsion angle restraints for these nucleotides. These helped obtain a well-defined loop with an average RMSD of 1.1 A for the loop nucleotides of 11 converged structures.
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PMID:NMR structure of a bacteriophage T4 RNA hairpin involved in translational repression. 867 67

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.
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PMID:DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity. 867 29

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.
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PMID:DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site. 879 44

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein. Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.
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PMID:Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57. 905 69

Transfer RNA(Lys)SUU, with a 5-modified-2-thiouridine at wobble position 34, facilitates -1 frameshifts for correct translation of the Escherichia coli DNA polymerase gamma subunit and retroviral polymerases. Peptidyl-tRNA(Lys)SUU prematurely terminates translation more often than other tRNAs. In order to determine if the anticodon structures of bacterial and mammalian tRNA(Lys)SUU species explain these observations, oligonucleotides corresponding to the anticodon regions of mammalian and E. coli tRNA(Lys)SUU were synthesized and their physicochemical properties compared with that of E. coli tRNA(Glu)SUC. The anticodon region of tRNA(Lys)SUU was stabilized by an unusual interaction between the side chains of the 5-modified-s(2)U34 and N-6-threonylcarbamoyl-adenosine-37 (t(6)A37), a combination of modified nucleosides unique to tRNA(Lys)SUU species. This first observation of modified nucleoside side-chain interactions is analogous to the interactions of amino acid side chains in proteins. The tRNA(Lys)SUU anticodon structure was determined from NMR restraints on model oligonucleotides. With only two of three anticodon bases available for codon pairing, this unconventional anticodon structure is a reasonable explanation for the bacterial and mammalian tRNA(Lys)SUU tendency to frameshift. A two-out-of-three reading of coding triplets also explains the increased rate at which peptidyl-tRNA(Lys)SUU prematurely terminates translation. In addition, modified nucleoside interaction distorts the anticodon loop. The distorted loop is a possible structural determinant for the preferential selection of tRNA(Lys3)SUU as primer of HIV-1 reverse transcriptase in vivo.
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PMID:Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1. 908 48


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