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 DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.
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PMID:Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data. 1177 7

As part of a high-throughput, structural proteomic project we have used NMR spectroscopy to determine the solution structure and ascertain the function of a previously unknown, conserved protein (MtH895) from the thermophilic archeon Methanobacterium thermoautotrophicum. Our findings indicate that MtH895 contains a central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other. It has an overall fold superficially similar to that of a glutaredoxin. However, detailed analysis of its three-dimensional structure along with molecular docking simulations of its interaction with T7 DNA polymerase (a thioredoxin-specific substrate) and comparisons with other known members of the thioredoxin/glutaredoxin family of proteins strongly suggest that MtH895 is more akin to a thioredoxin. Furthermore, measurement of the pK(a) values of its active site thiols along with direct measurements of the thioredoxin/glutaredoxin activity has confirmed that MtH895 is, indeed, a thioredoxin and exhibits no glutaredoxin activity. We have also identified a group of previously unknown proteins from several other archaebacteria that have significant (34-44%) sequence identity with MtH895. These proteins have unusual active site -CXXC- motifs not found in any known thioredoxin or glutaredoxin. On the basis of the results presented here, we predict that these small proteins are all members of a new class of truncated thioredoxins.
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PMID:Identification of a novel archaebacterial thioredoxin: determination of function through structure. 1193 70

The G --> T transversion is the dominant mutation induced by the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct. The structure of d(ACATC(AFB)GATCT).d(AGATAGATGT), in which the cationic adduct was mismatched with deoxyadenosine, was refined using molecular dynamics calculations restrained by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained molecular dynamics calculations refined structures with pairwise rmsd <1 A and a sixth root R1x factor between the refined structure and NOE data of 10.5 x 10-2. The mismatched duplex existed in a single conformation at neutral pH. The aflatoxin moiety intercalated above the 5' face of the modified (AFB)G. The mismatched dA was in the anti conformation about the glycosyl bond. It extruded toward the major groove and did not participate in hydrogen bonding with (AFB)G. The structure was compared with that of d(ACATCGATCT).d(AGATAGATGT) containing the corresponding unmodified G.A mismatch and with d(ACATC(AFB)GATCT).d(AGATCGATGT) containing the aflatoxin lesion in the correctly paired (AFB)G.C context. The correctly paired oligodeoxynucleotide exhibited Watson-Crick-type geometry at the (AFB)G.C pair. It melted at higher temperature than the mismatched (AFB)G.A duplex. The unmodified mismatched G.A duplex exhibited spectral line broadening at neutral pH, suggesting a mixture of conformations. It exhibited a lower melting temperature than did the mismatched (AFB)G.A duplex. These differences correlated with replication bypass experiments performed in vitro utilizing DNA polymerase I exo- [Johnston, D. S., and Stone, M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164]. Those experiments showed that correct insertion of dC opposite (AFB)G blocked replication by the enzyme, whereas incorrect insertion of dA opposite (AFB)G allowed full-length replication of the adducted template strand.
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PMID:Mispairing of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct with deoxyadenosine results in extrusion of the mismatched dA toward the major groove. 1196 7

Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.
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PMID:Mapping of the interaction interface of DNA polymerase beta with XRCC1. 1246 78

Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase beta (beta-Pol). Here we report a site- directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to beta-Pol. Mutant proteins defective in XRCC1-NTD interaction with beta-Pol and with a beta-Pol-gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass beta-strand D and E of the five-stranded beta-sheet (betaABGDE) and the protruding alpha2 helix of the XRCC1-NTD. Mutations that included F67A (betaD), E69K (betaD), V86R (betaE) on the five-stranded beta-sheet and deletion of the alpha2 helix, but not mutations within alpha2, abolished binding of the XRCC1-NTD to beta-Pol. A Y136A mutant abolished beta-Pol binding, and a R109S mutant reduced beta-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and delta-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the alpha2 helix and the betaD and betaE strands of XRCC1-NTD to the energetics of beta-Pol binding. Establishing the direct contacts in the beta-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.
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PMID:Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase beta. 1252 65

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (epsilon) subunit of HE provides the 3'-->5' exonucleolytic proofreading activity for this complex. Epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). In addition to alpha, epsilon also binds the small (8 kDa) theta (theta) subunit. The function of theta is unknown, although it has been hypothesized to enhance the 3'-->5' exonucleolytic proofreading activity of epsilon. Using NMR analysis and molecular modeling, we have previously reported a structural model of epsilon186, the N-terminal catalytic domain of epsilon [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and C(beta) resonances of [U-(2)H,(13)C,(15)N] methyl protonated epsilon186 in complex with unlabeled theta. A structural comparison of the epsilon186-theta complex with free epsilon186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound epsilon186 revealed a potential binding surface on epsilon for interaction with theta involving structural elements near the epsilon catalytic site. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1 and beta-strands 2 and 3 and to the region near the beginning of alpha-helix 7. Additionally, a small stretch of residues (K158-L161), which previously had not been assigned in uncomplexed epsilon186, is predicted to adopt beta-strand secondary structure in the epsilon186-theta complex and may be significant for interaction with theta. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of epsilon may be stabilized by binding theta.
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PMID:Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy. 1266 53

An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a specific pairing partner of 9-methylimidazo[(4,5)-b]pyridine (Q). The Q base is known to pair with 2,4-difluorotoluene (F) as an isostere of the A-T pair, and F also pairs with A efficiently in replication. In contrast, the Q-Pa pair showed specific selectivity in replication, and the five-membered-ring base Pa paired efficiently with Q but paired poorly with A. In addition, the interaction of Pa with DNA polymerases was superior, in comparison to that of F. The aldehyde group of Pa was recognized well by the Klenow fragment of Escherichia coli DNA polymerase I and the reverse transcriptase of Avian myeloblastosis virus. The structural features of the Q-Pa pair in a DNA duplex were analyzed by NMR, showing the shape complementarity of the Pa fitting with Q. The structurally unique base Pa provides valuable information for the development of unnatural base pairs toward the expansion of the genetic alphabet.
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PMID:An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo[(4,5)-b]pyridine. 1272 Apr 41

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.
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PMID:Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. 1276 57

Nucleotides 2-(4-azidophenacyl)thio-1,N6-etheno-2'-deoxyadenosine 5'-triphosphate 1 and its tetrafluoro analog 2 inhibit HIV-1 reverse transcriptase (RT) competitively relative to template. These template-competitive RT inhibitors (TCRTIs) were analyzed for conformational properties by molecular modeling and NMR analysis. Both inhibitors prefer sugar conformations of C2'-endo/C3'-exo with a high-anti glycosidic bond rotation and +sc/ap phosphate conformation (gamma). The major effect of the etheno group is to favor an extended, fully staggered anti conformation in the N1-C2-S-CH2 psi1 side chain rotation, and NMR analysis detects a long range sugar H4' to side chain phenyl meta-H NOE, a result consistent with this compact structure as an important contributor to the solution structure. The binding model generated places the phenyl side chain in a lipophilic pocket in the template grip region of the RT polymerase domain with the Mg-triphosphate complexed to active site carboxylates. The structures of the TCRTIs are compared with that of the template-competitive DNA polymerase inhibitor 2-(4-azidophenacyl)thio-2'-deoxyadenosine 5'-triphosphate 3, and a theoretical model for selectivity is proposed.
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PMID:Conformational properties of nucleotide-based template-competitive HIV-1 reverse transcriptase inhibitors: analysis of enzyme binding modes. 1281 87

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes.
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PMID:Solution structure of a wedge-shaped synthetic molecule at a two-base bulge site in DNA. 1285 93


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