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)

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.
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PMID:Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. 751 81

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
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PMID:Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus. 754 60

We have examined the biophysical properties of DNA polymerase beta (beta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide (kq = 1.59 x 10(9)M-1S-1), and by a negatively charged ionic quencher, I- (kq = 1.60 x 10(9) M-1S-1), but not by a positively charged ionic quencher, Cs+ (kq = 0.2 x 10(9) M-1S-1). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (> 0.05 M) studied in the absence of divalent metals. Zn2+ causes higher order aggregation, but no such aggregates are seen with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs+) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S0(20),W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.
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PMID:Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase beta. 796 32

Mutations in one C1 INH allele result in the autosomal dominant disease, hereditary angioedema. The plasma antigenic level of C1 INH in this disease may be low, normal, or high, while the functional level is uniformly depressed. Investigation of the mutations in the C1 INH gene reveal several key features about the DNA itself as well as protein structure-function relationships. The largest single group of mutations with a defined mechanism are recombinations associated with Alu repetitive DNA elements. Current data suggest that there may be an increased number of mutations within the region encoding the reactive center which, like some other serpins, contains both primary and secondary structure DNA polymerase pause sites. These sites may enhance the rates of mutation and evolution in the reactive center region. Some of the dysfunctional C1 INH proteins that result from hinge region mutations support models for reactive center loop interaction with beta sheet A during complex formation. The analysis of the dysfunctional mutants, therefore, suggest regions of the molecule that are important for inhibitor function.
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PMID:Mutations in the C1 inhibitor gene that result in hereditary angioneurotic edema. 817 83

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.
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PMID:Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity. 965 93

We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.
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PMID:Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis. 1098 85

The DNA polymerase (pol) catalytic subunit of herpes simplex virus type 1, encoded by UL30, and its accessory factor, UL42 protein, are both essential for the replication of the virus. Because the stable interaction between UL42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the development of novel antiviral compounds. To better compare the effects of mutations in UL42 protein on its known in vitro functions, mutations were expressed as glutathione-S-transferase (GST)-fusions and the fusion proteins used in affinity chromatography. In this report, we demonstrate the relationship between the abilities of mutant UL42 fusion proteins to bind pol and to stimulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusion proteins was found to be a more accurate and sensitive measure of the ability of the UL42 protein to function in vitro than the pol binding assay using the fusion proteins linked to a solid matrix. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells. Our results demonstrate that two noncontiguous stretches of amino acids, from 137 to 142 and from 274 to 282, are essential for UL42 function in vivo and in vitro. Although mutant d241-261 exhibited close to wild-type abilities to stimulate pol activity in vitro, it was not capable of complementing the replication of a UL42 null mutant virus. The region of UL42 protein within or close to 241-261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothesized that reduction in the length of the hinge region could alter the ability of UL42, and/or its complex with pol, to function with one or more of the other proteins present in the DNA replisome within infected cells.
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PMID:Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function. 1099 37

The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau protein. Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance. Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V).
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PMID:tau binds and organizes Escherichia coli replication through distinct domains. Partial proteolysis of terminally tagged tau to determine candidate domains and to assign domain V as the alpha binding domain. 1107 43

The highly conserved GXD sequence present in the Mycobacterium tuberculosis DNA polymerase I corresponds to a hinge region in the finger subdomain connecting M and N helices of Escherichia coli pol I. An examination of the crystal structures of pol I family polymerases reveals that the invariant aspartate of the hinge forms a salt bridge with the conserved arginine of the O-helix and an H-bond with Gln-708. To clarify the role of this region, we generated and characterized conserved and nonconserved mutant derivatives of this aspartate, the preceding glutamate and the Gln in TB pol I. For comparison, D732A mutein of pol I was also included. The muteins representing conserved aspartate (Asp-707 of TB pol I or Asp-732 of pol I) showed a strong K(m)((dNTP)) effect and minor alteration in K(d)((DNA)), with about 10-20-fold decrease in overall catalytic efficiency. The TB muteins, E706A and Q683A, have less pronounced deviations from the wild-type enzyme. Further examination of D707A of TB pol I showed no alteration in the processivity or the dideoxynucleotide sensitivity patterns. However, both TB pol D707A and homologous E. coli D732A failed to form a stable E.DNA.dNTP ternary complex. These results suggest that the aspartate in the hinge region is catalytically important and is required for dNTP binding and in the formation of a prepolymerase ternary complex.
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PMID:DNA polymerase I of Mycobacterium tuberculosis: functional role of a conserved aspartate in the hinge joining the M and N helices. 1167 39

DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.
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PMID:Variants of DNA polymerase Beta extend mispaired DNA due to increased affinity for nucleotide substrate. 1296 95


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