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)

Levodopa and dopamine are naturally occurring catecholamines with antitumor activity in several experimental tumor systems. Previous studies suggested that their cytotoxic effect was related in part to their inhibitory effect upon DNA polymerase. We have examined the effects of levodopa, dopamine, levodopa methyl ester, norepinephrine, and the analog 3,4-dihydroxybenzylamine upon human and murine melanoma cells. When exponentially growing cells were exposed to these drugs, a characteristic inhibition of thymidine incorporation was observed with much less inhibition of either uridine or leucine incorporation. In order to ascertain that inhibition was occurring at the level of DNA synthesis, we examined the effects of the drugs upon the incorporation of thymidine triphosphate by permeabilized melanoma cells. When melanoma cells were permeabilized by lysolecithin, thereby permitting the direct incorporation of labeled thymidine triphosphate, a similar inhibition of incorporation was observed. Dopamine at a concentration of 4.8 microM caused a 50% reduction in incorporation of label. These results suggested that inhibition did occur at the level of DNA synthesis. In the presence of the melanocyte-specific oxidase, tyrosinase, these derivatives are potent inhibitors of isolated DNA polymerase alpha with 50% inhibitory concentrations between 1 and 10 microM. The inhibition could be completely prevented by the presence of reducing agents such as dithiothreitol (1.0 mM). The quinols themselves were not inhibitors of DNA polymerase. Dopamine analogs represent an interesting class of antitumor agents with inhibitory activity for DNA polymerase.
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PMID:Levodopa and dopamine analogs as DNA polymerase inhibitors and antitumor agents in human melanoma. 676 47

mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.
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PMID:Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region. 694 48

We have studied selected mutants of human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT) in a cell-free system in order to assess whether the mutant proteins exhibit a reduction in the sensitivity to nucleoside analog inhibitors similar to that of HIV-1 RT. We have modified, by site-directed mutagenesis, several of those amino acid residues so that their equivalent substitutions in HIV-1 RT have led to the formation of HIV-1 RT variants with the highest degree of resistance to dideoxynucleoside triphosphates (i.e., Glu-89-->Gly, Leu-74-->Val, and Ser-215-->Tyr [which is comparable to the Thr-215-->Tyr mutation of HIV-1 RT] and the double mutations Glu-89-->Gly/Ser-215-->Tyr and and Leu-74-->Val/Ser-215-->Tyr). The similarity found between resistance of the newly generated HIV-2 RT mutants to nucleoside analogs and that of the comparable mutants of HIV-1 RT can support the notion that the overall protein folding around the DNA polymerase active site in HIV-2 RT is quite similar to that of HIV-1 RT.
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PMID:Resistance to nucleoside analogs of selective mutants of human immunodeficiency virus type 2 reverse transcriptase. 752 86

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
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PMID:Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. 752 32

As the sole renal Na,K-ATPase isozyme, the alpha 1 Na,K-ATPase accounts for all active transport of Na+ throughout the nephron. This role in renal Na+ reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)1079-->thymine (T) transversion, resulting in the substitution of glutamine276 with leucine and associated with decreased net 86Rb+ (K+) influx, was identified in Dahl salt-sensitive/JR rat kidney alpha 1 Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T1079 but rather only the wild-type A1079 alpha 1 Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined alpha 1 Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A1079-->T transversion in the Dahl salt-sensitive alpha 1 Na,K-ATPase allele and its transcript, as well as the wild-type A1079 sequence in the Dahl salt-resistant alpha 1 Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T1079 alpha 1 cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type alpha 1 Na,K-ATPase sequence. This identifies a site- and nucleotide-specific Taq polymerase misincorporation, suggesting that a structural basis might underlie a predisposition to nonrandom Taq polymerase errors.
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PMID:Confirmation of mutant alpha 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats. 808 31

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.
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PMID:Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development. 808 46

Peptide I, a 50-amino acid synthetic peptide based on residues 728 to 777 of DNA polymerase I, binds dNTP substrates and duplex DNA (G. Mullen, P. Shenbagamurthi, and A.S. Mildvan, J. Biol. Chem. 264, 19637-19647, 1988). The structural properties of peptide I at pH 3.9 have been studied by CD spectroscopy and by 2D proton NMR at 600 MHz. The CD spectra are fit by assuming that peptide I contains 17% helix, 17% beta-structure, and 66% coil. The substrate dATP binds tightly to peptide I under these conditions (KD = 0.5 microM) as determined by fluorescence quenching but induces no change in peptide conformation, as detected by CD spectroscopy. Proton resonances of peptide I have been assigned by double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy. As found with other peptides, peptide I is best characterized by both extended and partially folded secondary structures which equilibrate rapidly on the NMR time scale. A region from residues 3 through 10 displays nuclear Overhauser effects (NOEs) consistent with the rapid equilibration of a nascent helix with a random extended structure. Alternatively this segment of residues is consistent with a series of three opened-out turns. A nonclassical turn is found between residues 14 and 17 and from residues 44 to 47, the latter closing irregular antiparallel strands from residues 42 to 48. The remainder of the peptide is a coil. A residue-by-residue comparison of the best-fit solution structure of the peptide with that of the corresponding sequence in the X-ray structure of the complete enzyme reveals that 36% of the amino acids are found to be in a conformation similar to that in the enzyme. Such partial and transient folding of the peptide indicates that the major role of the remainder of the protein is to provide structural support for the active site region of the enzyme. As detected by interresidue NOEs and NOEs to water protons, the homologous sequence Leu-37-Ile-38-Tyr-39-Gly-40, together with Phe-15 of the peptide, provides an exposed hydrophobic cluster of residues which may constitute the substrate binding site. An exposed cluster of cationic residues consisting of Arg-27, Arg-28, Lys-31, and possibly Arg-48 may provide the binding site for duplex DNA.
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PMID:Sequential proton NMR resonance assignments, circular dichroism, and structural properties of a 50-residue substrate-binding peptide from DNA polymerase I. 844 59

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.
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PMID:Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta. 864 5

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.
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PMID:Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis. 876 85

Both ganciclovir-sensitive and -resistant human cytomegaloviruses (HCMV) were isolated from a patient with aplastic anemia complicated with CMV retinitis and encephalitis. Ganciclovir-resistant clinical isolate, 93-1R, also showed cross-resistance against (s)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir). Molecular analysis of plaque-cloned strains revealed that a single nucleotide substitution at 2160 (C to T) resulted in amino acid substitution at codon 501 from leucine to phenylalanine in the DNA polymerase gene. This mutation at codon 501 was easily identified by means of AluI digestion of the selected PCR product. The same mutation existed in the DNA fragment amplified from the patient's brain, suggesting that cross-resistant mutant 93-1R caused encephalitis. Furthermore, ganciclovir-resistant 93-1R-3 replicated much faster and was released more efficiently into the culture medium than ganciclovir-sensitive 91-7S-1.
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PMID:Genetic analysis of a clinical isolate of human cytomegalovirus exhibiting resistance against both ganciclovir and cidofovir. 912 39


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