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 polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol beta. Since the chemical step may not be rate-limiting for wild-type pol beta, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol beta displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.
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PMID:DNA polymerase beta: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics. 917 67

The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.
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PMID:Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5'-deoxyribose phosphate lyase activities. 955 98

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.
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PMID:Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies. 959 46

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.
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PMID:Probing the binding region of the single-stranded DNA-binding domain of rat DNA polymerase beta using nanosecond-pulse laser-induced cross-linking and mass spectrometry. 974 86

A novel class of DNA-binding domains has been established from at least sixteen recently identified DNA-binding proteins. The three-dimensional structure of one of these domains, Mrf-2, has been solved using NMR methods. This structure is significantly different from known DNA-binding domain structures. The mechanism of DNA recognition by this motif has been suggested based on conserved residues, surface electrostatic potentials and chemical shift changes. This new DNA-binding motif shares structural homology with T4 RNase H, E. coli endonuclease III and Bacillus subtilis DNA polymerase I. The structural homology suggests a mechanism for substrate recognition by these enzymes.
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PMID:A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases. 980 40

Streptomyces sp. KM1-30 was isolated from soil as a producer of antimutagens by screening with a modified Ames test. The chemical structure of the antimutagenic metabolite was identified as streptovaricin C, which is known to inhibit DNA dependent RNA polymerase from E. coli and RNA dependent DNA polymerase from RNA tumor viruses, by MS and 1H-, 13C-NMR analyses. Addition of streptovaricin C to the cultures of UV treated Salmonella typhimurium TA100 or Trp-P-2-treated S. typhimurium TA98 decreased the frequency of mutation without a decrease in viable cell counts. The effect of streptovaricin C to the mutation induced by UV and Trp-P-2 was not desmutagenic, but antimutagenic.
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PMID:An antimutagenic metabolite, streptovaricin C, isolated from Streptomyces sp. 998 75

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.
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PMID:A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit. 1037 10

We investigated the behaviour of a 15mer DNA duplex, [5'd(CAGAGTCACTGGCTC)3']. [5'd(GAGCCAG)3' + 5'd(GACTCTG)3'] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pKa of approximately 4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G.C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G.A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn).A+(anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three-dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.
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PMID:Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide). An NMR study and molecular dynamic simulations with explicit water molecules. 1044 80

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C(18)-linoleic acid (LA) or C(24)-nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an Omega-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K(D)) of 1.02 or 2.64 mM, respectively.
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PMID:Mode analysis of a fatty acid molecule binding to the N-terminal 8-kDa domain of DNA polymerase beta. A 1:1 complex and binding surface. 1046 95

The mammalian X-ray cross-complementing group 1 protein (XRCC1) is an important player in base excision repair of damaged DNA. Two new findings help to elucidate its role - biochemical data suggest that this multidomain protein interacts not only with three different enzymes, but also with the nicked DNA itself, and NMR data reveal the structure of the domain that interacts with both DNA polymerase beta and DNA.
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PMID:Holding damaged DNA together. 1046 2


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