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 quinone intermediates resulting from tyrosinase-mediated oxidation of tyrosine were evaluated as sulfhydryl reagent inhibitors of purified calf thymus DNA polymerase alpha in order to determine which of these might be cytotoxic. Dopachrome and an oxidation product of 2,4,5-trihydroxyphenylalanine were relatively ineffective as inhibitors of DNA polymerase alpha. On the other hand, a dopaquinone analogue, 4-(2-N-acetylaminoethyl)-1,2-benzoquinone, synthesized from N-acetyl dopamine, was demonstrated to have marked affinity for this sulfhydryl enzyme. This property was shared by 1,2-benzoquinone. These studies point to dopaquinone as a significant toxic metabolite in melanin biosynthesis.
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PMID:The toxicity of melanin precursors. 41 70

The sequence of the first 29 nucleotides in the promoter region of a tyrosine tRNA gene has previously been determined (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). This work has now been extended to give the sequence of a total of 59 nucleotides; the sequence is as follows: (see article). The general approach used in the determination of the sequence involved the DNA polymerase I-catalyzed elongation of synthetic deoxyribopolynucleotide primers hydridized to the l-strand of phi80psu+III DNA at the appropriate site. Sequencing of the newly added nucleotides was facilitated by the use of a number of techniques including (a) elongation of the primer with the use of all of the four nucleoside 5'-triphosphates but limiting the concentration of one of the triphosphates, (b) insertion of ribonucleotide units at appropriate sites so as to permit subsequent specific cleavages by pancreatic RNase, and (c) two-dimensional fingerprinting of the oligonucleotides in conjunction with partial exonucleolytic degradation, comprehensive nearest neighbor analyses, and the determination of pyrimidine tracts.
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PMID:The nucleotide sequence in the promoter region of the gene for an Escherichia coli tyrosine transfer ribonucleic acid. 78 Mar 52

Regenerating rat liver was used as a semisynchronous system in which to investigate the effects of 6-thioguanine on biochemical processes occurring in discrete phases of the cell cycle. 6-Thioguanine inhibited the first wave of DNA biosynthesis in regenerating rat liver. This effect appeared to be the result of a decrease, caused by 6-thioguanine, in the induction of several enzyme activities (i.e., thymidine kinase, deoxycytidylate deaminase, cytidine diphosphate reductase, and DNA polymerase) necessary for the initiation of DNA replication in regenerating liver. There was a fairly short period during which 6-thioguanine could be given to rats to accomplish the inhibition of the appearance of the induced activities of these enzymes; this period corresponded to the time just before enzyme induction. The inhibition of the induced synthesis of this group of enzymes occurred in the presence of an intact translational apparatus and intact polysomes and in the absence of interference with the incorporation of radioactive leucine and tyrosine into total protein of liver. Synthesis of polyadenylate-containing RNA was depressed in 6-thioguanine-treated rats, whereas the synthesis of polyadenylate-lacking RNA was unaffected. It is suggested that the inhibition of the synthesis of polyadenylate-containing RNA by 6-thioguanine is at least in part responsible for the observed decrease in induced enzyme activities and the resulting interference with DNA replication.
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PMID:Effects of 6-thioguanine on macromolecular events in regenerating rat liver. 87 Jan 91

The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined. This is: (5') ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5'). The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site. Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts. Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.
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PMID:The nucleotide sequence in the promoter region of the fene for an Escherichia coli tyrosine transfer ribonucleic acid. 108 50

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.
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PMID:Structural and functional studies on phi 29 DNA polymerase. 129 Dec 40

It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations (Asn-67, Arg-70, and Tyr-215). Construction of a molecular clone with this genotype gave rise to only a partially resistant virus, raising the possibility that an additional mutation existed in some clinical isolates. Using an HIV marker rescue system, we have mapped and identified a fifth mutation conferring resistance to zidovudine, namely, methionine to leucine at codon 41 of HIV RT. An infectious molecular clone containing this mutation together with three previously identified mutations in the RT coding sequence yielded highly resistant HIV after transfection of T cells. Direct detection of the fifth mutation in DNA samples from cocultured peripheral blood lymphocytes by the PCR revealed that it occurred relatively early in the development of zidovudine resistance. However, this mutation was only detected after the appearance of the codon 215 change in the RT coding sequence. Identification of this mutation in addition to the other known mutations conferring resistance enables rapid and direct correlation between an RT genotype and sensitivity of the virus.
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PMID:Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine. 137 86

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.
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PMID:Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities. 139 3

The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.
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PMID:Genetic organization and structural features of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa. 142 26

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.
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PMID:[Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides]. 188 93

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
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PMID:Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids. 200 32


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