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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.
...
PMID:The hydrophobic hinge region of rat DNA polymerase beta is critical for substrate binding pocket geometry. 1590 25

Thirty percent of the 189 tumors studied to date express DNA polymerase beta variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein. Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorage-independent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase beta variant proteins, implying that it has a mutational basis. Because DNA polymerase beta functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.
...
PMID:Expression of DNA polymerase {beta} cancer-associated variants in mouse cells results in cellular transformation. 1617 90

Eukaryotic DNA polymerase (Pol) delta replicates chromosomal DNA and is also involved in DNA repair and genetic recombination. Motif A in Pol delta, containing the sequence DXXXLYPSI, includes a catalytically essential aspartic acid as well as other conserved residues of unknown function. Here, we used site-directed mutagenesis to create all 19 amino acid substitutions for the conserved Leu(612) in Motif A of Saccharomyces cerevisiae Pol delta. We show that substitutions at Leu(612) differentially affect viability, sensitivity to genotoxic agents, cell cycle progression, and replication fidelity. The eight viable mutants contained Ile, Val, Thr, Met, Phe, Lys, Asn, or Gly substitutions. Individual substitutions varied greatly in the nature and extent of attendant phenotypic deficiencies, exhibiting mutation rates that ranged from near wild type to a 37-fold increase. The L612M mutant exhibited a 7-fold elevation of mutation rate but essentially no detectable effects on other phenotypes monitored; the L612T mutant showed a nearly wild type mutation rate together with marked hypersensitivity to genotoxic agents; and the L612G and L612N strains exhibited relatively high mutation rates and severe deficits overall. We compare our results with those for homologous substitutions in prokaryotic and eukaryotic DNA polymerases and discuss the implications of our findings for the role of Leu(612) in replication fidelity.
...
PMID:Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase delta. 1634 51

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile(135) of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.
...
PMID:Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44. 1637 49

The nature of conformational transitions in DNA polymerase lambda (pol lambda), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol lambda has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg(2+) ion in the active site, and with alterations in active site residues Ile(492) and Arg(517). Though flips occurred for several side-chain residues (Ile(492), Tyr(505), Phe(506)) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing beta-strand 8 in the thumb, but closing occurred only in the Ile(492)Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile(492)Ala and Arg(517)Ala mutant studies. Our combined studies suggest that while pol lambda does not demonstrate large-scale subdomain movements as DNA polymerase beta (pol beta), significant DNA motion exists, and there are sequential subtle side chain and other motions-associated with Arg(514), Arg(517), Ile(492), Phe(506), Tyr(505), the DNA, and again Arg(514) and Arg(517)-all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol lambda to the chemistry-competent state. Significantly, analogs of these residues in pol beta (Lys(280), Arg(283), Arg(258), Phe(272), and Tyr(271), respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol beta, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.
...
PMID:Sequential side-chain residue motions transform the binary into the ternary state of DNA polymerase lambda. 1692 Aug 35

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic HBV infection may progress to cirrhosis and hepatocellular carcinoma. Mutations at the active site of DNA polymerase of HBV, tyrosine-methionine-aspartate-aspartate (YMDD) motif, render infected patients resistant to antiviral drug (Lamivudine) therapy. Hence, sensitive and specific methods aimed at detecting the mutants are essential. The purpose of this study was to develop methods for detecting the mutations at YMDD by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR using locked nucleic acid (LNA)-mediated TaqMan probes. The results obtained by these methods were compared with those examined by conventional direct sequencing on serum samples of 77 patients treated with lamivudine. Our results show that both PCR-RFLP and real-time PCR could detect wild type, YMDD, and its mutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valine-aspartate-aspartate. In addition, the mixtures of the wild-type virus and its mutants in the serum sample were detected. Importantly, real-time PCR is less time-consuming, and more sensitive for the detection of mixed populations than PCR-RFLP. The real-time PCR with LNA-mediated TaqMan probes is a sensitive, specific and rapid detection method for mutations at the YMDD motif, which will be essential for monitoring patients undergoing lamivudine antiviral therapy.
...
PMID:Rapid detection of lamivudine-resistant hepatitis B virus mutations by PCR-based methods. 1696 Mar 47

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.
...
PMID:Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage. 1821 33

Y-family DNA polymerases (DNAPs) are often required in cells to synthesize past DNA-containing lesions, such as [+ta]-B[a]P-N(2)-dG, which is the major adduct of the potent mutagen/carcinogen benzo[a]pyrene. The current model for the non-mutagenic pathway in Escherichia coli involves DNAP IV inserting deoxycytidine triphosphate opposite [+ta]-B[a]P-N(2)-dG and DNAP V doing the next step(s), extension. We are investigating what structural differences in these related Y-family DNAPs dictate their functional differences. X-ray structures of Y-family DNAPs reveal a number of interesting features in the vicinity of the active site, including (1) the "roof-amino acid" (roof-aa), which is the amino acid that lies above the nucleobase of the deoxynucleotide triphosphate (dNTP) and is expected to play a role in dNTP insertion efficiency, and (2) a cluster of three amino acids, including the roof-aa, which anchors the base of a loop, whose detailed structure dictates several important mechanistic functions. Since no X-ray structures existed for UmuC (the polymerase subunit of DNAP V) or DNAP IV, we previously built molecular models. Herein, we test the accuracy of our UmuC(V) model by investigating how amino acid replacement mutants affect lesion bypass efficiency. A ssM13 vector containing a single [+ta]-B[a]P-N(2)-dG is transformed into E. coli carrying mutations at I38, which is the roof-aa in our UmuC(V) model, and output progeny vector yield is monitored as a measure of the relative efficiency of the non-mutagenic pathway. Findings show that (1) the roof-aa is almost certainly I38, whose beta-carbon branching R-group is key for optimal activity, and (2) I38/A39/V29 form a hydrophobic cluster that anchors an important mechanistic loop, aa29-39. In addition, bypass efficiency is significantly lower both for the I38A mutation of the roof-aa and for the adjacent A39T mutation; however, the I38A/A39T double mutant is almost as active as wild-type UmuC(V), which probably reflects the following. Y-family DNAPs fall into several classes with respect to the [roof-aa/next amino acid]: one class has [isoleucine/alanine] and includes UmuC(V) and DNAP eta (from many species), while the second class has [alanine (or serine)/threonine] and includes DNAP IV, DNAP kappa (from many species), and Dpo4. Thus, the high activity of the I38A/A39T double mutant probably arises because UmuC(V) was converted from the V/eta class to the IV/kappa class with respect to the [roof-aa/next amino acid]. Structural and mechanistic aspects of these two classes of Y-family DNAPs are discussed.
...
PMID:Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli. 1960 44

The high yield and specificity of PCR amplifications are affected by DNA polymerase activity at room temperature. One way of preventing this unwanted activity is by genetic modifications of the DNA polymerase. For Taq DNA polymerase, mutations in the gene (Glu626Lys, Trp706Arg, Ile707Leu and Glu708Asp), when introduced individually or in certain combinations, were found to contribute to a significant decrease of the enzyme activity at room temperature. The aim of this study was to evaluate the usefulness of the Ile707Leu cold-sensitive mutation in the N-terminal deletional variant of Taq DNA polymerase in PCR reaction. The Ile(707) to Leu substitution was introduced to Klentaq278 by site-directed mutagenesis. Normal and mutant DNA polymerases were expressed under a tac promoter and purified to homogeneity. The mutant polymerase showed reduced polymerase activity at room temperature by up to 12 times and no significant change in thermostability, compared to Klentaq278 DNA polymerase. The major effect of the amino acid substitution was the reduction of the amplification capacity of the polymerase. Mutant polymerase could not amplify fragments over 1 kb. In conclusion, the substitution of Ile707Leu in Klentaq278 DNA polymerase reduces the overall processivity of the enzyme and therefore limits the application of this DNA polymerase in PCR.
...
PMID:Substitution of Ile(707) for Leu in Klentaq DNA polymerase reduces the amplification capacity of the enzyme. 2008 49

Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.
...
PMID:Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea: features and phylogeny. 2274 67


<< Previous 1 2 3 4 5 Next >>

---