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 sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.
...
PMID:Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding. 203 95

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.
...
PMID:Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase. 211 23

Two unstable hemoglobins (Hbs) causing rather severe hemolytic anemia have been characterized. The beta chain of Hb Birmingham, found in an adult black man, is characterized by the loss of -Leu-Ala-His-Lys- at positions 141, 142, 143, and 144 and their replacement by one Gln residue. These changes are the result of a deletion of nine nucleotides, namely two base pairs (bp) of codon 141, all of codons 142 and 143, and one bp of codon 144; the remaining CAG triplet (C from codon 141 and AG from codon 144) codes for the inserted glutamine. In the beta chain of Hb Galicia from a Spanish patient, His and Val at positions 97 and 98 are replaced by one Leu residue. This is due to an ACG deletion in codons 97 and 98, which causes the removal of one His and one Val residue, while the remaining CTG triplet (C from codon 97 and TG from codon 98) codes for the inserted leucine residue. Two mechanisms, namely slipped mispairing in the presence of short repeats, and misreading by DNA polymerase due to a local distortion of the DNA helix, are considered in explaining the origin of the small deletions.
...
PMID:Hemoglobin Birmingham and hemoglobin Galicia: two unstable beta chain variants characterized by small deletions and insertions. 215 27

By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction. Replacement of Phe-181 by alanine reduced the enzyme activity only 30%. Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively. The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate. Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold. Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta.
...
PMID:Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition. 219 36

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment. 220 84

Insulin resistance is an early predictor of development of noninsulin-dependent diabetes mellitus (NIDDM) in Pima Indians, a population with the highest reported prevalence of NIDDM. The insulin receptor plays a central role in mediating insulin action, and previous studies have demonstrated that mutations in the insulin receptor gene may cause insulin resistance. Therefore, we have cloned the insulin receptor cDNA from an insulin-resistant Pima Indian to determine if there is a mutation in the patient's insulin receptor gene. We obtained nine cDNA clones spanning exons 4-10 and 12-22 of the patient's insulin receptor gene. Polymorphisms in the nucleotide sequences for codons 523 (Ala), 1058 (His), and 1062 (Leu) provided useful markers to differentiate the patient's two alleles of the insulin receptor gene. These substitutions were silent, in that they did not alter the predicted amino acid sequence. The sequence of exons 1-3 and 11 was determined directly from genomic DNA that had been amplified using the polymerase chain reaction catalyzed by Taq DNA polymerase. Other investigators have reported defects in insulin binding and insulin receptor tyrosine kinase activity in diabetic Pima Indians. However, we did not detect any mutations in this patient's insulin receptor gene. Thus, these observations are consistent with the interpretation that the defects in insulin receptor function are acquired rather than derived from defects in the primary structure of the receptor.
...
PMID:The amino acid sequence of the insulin receptor is normal in an insulin-resistant Pima Indian. 231 37

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.
...
PMID:DNA polymerase III gene of Bacillus subtilis. 249 83

Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.
...
PMID:The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III. 251 95

The amino acid substitutions responsible for the temperature-sensitive (ts) and mutator phenotypes of the classical bacteriophage T4 DNA polymerase mutant tsL56 were determined. tsL56 DNA polymerase has two mutations in the 5' end of the DNA polymerase gene (g43) that produce two amino acid substitutions: codon 89, alanine to threonine, and codon 363, aspartate to asparagine. Both mutations are required for the strong ts and mutator phenotypes. The increased error rate of the tsL56 DNA polymerase is due to a reduction in 3'----5' exonuclease activity relative to polymerase activity (N. Muzyczka, R. L. Poland, and M. J. Bessman, J. Biol. Chem. 247:7116-7122, 1972). Thus, the locations of the tsL56 mutations suggest that the 3'----5' exonuclease domain resides in the N-terminal region. Several other ts DNA polymerase mutant strains isolated with tsL56 also have mutator or antimutator phenotypes. The nucleotide changes in these important mutant strains were also determined. This mutant collection, combined with collections of g43 amber mutants and mutants selected on the basis of a strong mutator phenotype (L. J. Reha-Krantz, J. Mol. Biol. 202:711-724, 1988), contains nearly 70 different DNA polymerase mutations. The numerous T4 DNA polymerase mutations are valuable for DNA polymerase structure-function and fidelity studies.
...
PMID:Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain. 267 3

By comparative sequence analysis of the herpes simplex virus type 1 DNA polymerase gene of strain Angelotti and a phosphonoacetic acid-resistant (PAAr) derivative, the site of the PAAr mutation was identified as a single nucleotide (C----T) conversion within the mapping limits of the known PAAr mutations of strains KOS and 17. The conservative amino acid change at residue 719 from alanine to valine results in a radical change in the properties of the polymerase, rendering the mutant enzyme resistant to PAA and various antiviral compounds. Amino acid homologies as well as secondary structure analysis reveal that the PAAr mutation is contained in a 14 amino acid sequence which is highly conserved, and detected in the central domain of prokaryotic and eukaryotic DNA polymerases.
...
PMID:The herpes simplex virus type 1 DNA polymerase gene: site of phosphonoacetic acid resistance mutation in strain Angelotti is highly conserved. 303 42


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---