Preferential digestion of (A plus T)-rich stretches of yeast mitochondrial DNA in isolated mitochondria.
(57/803)
Yeast mitochondrial DNA labelled in vitro by incubation of isolated mitochondria with DNA precursors exhibits skewed profiles on isopycnic CsCl gradients. The skew is not due to nuclear DNA nor to single-stranded mitochondrial DNA in the product labelled in vitro. Simultaneous labelling with [3H]dTTP and [14C]dGTP in vitro indicates a gradient of base composition in the DNA labelled in vitro. Thus, selective degradation of mitochondrial DNA occurs during incubation, converting large molecules having the mean density of mitochondrial DNA into smaller molecules of higher mean density and with higher G:T ratio. Similarly skewed distributions can also be produced by incubation of mitochondrial DNA labelled in vivo with the yeast mitochondrial fraction or with micrococcal endonuclease, an enzyme known to selectively hydrolyse (A plus T)-rich regions of DNA. (+info)
Ribonucleotide reductase, a possible agent in deoxyribonucleotide pool asymmetries induced by hypoxia.
(58/803)
While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension. (+info)
Misincorporation of (TP during transcription of poly dAT-dAT and poly dABU-dABU.
(59/803)
The synthetic DNA alternating copolymers poly dAT-dAT and dABU-dABU have been transcribed with E. coli RNA polymerase to measure the level of BrdU-induced misincorporation of guanine during transcription. GTP is found to be misincorporated into both copolymers at a frequency of 1 per 1000-2000 nucleotides polymerized. Using alpha-32P-GTP, the nearest neighbors to GMP are found to be UMP (approximately 63%), GMP (approximately 25%) and AMP (approximately 17%), with no apparent difference between the two templates. These results suggest that BrdU-substitution in DNA does not necessarily increase the potential for base mispairing during transcription, and hence, promote the production of faulty RNA molecules. (+info)
The use of primed synthesis by DNA polymerase I to study an intercistronic sequence of phiX-174 DNA.
(60/803)
A decadeoxynucleotide complementary to ten nucleotides in the major ribosome-protected fragment of phiX-174 plus-strand DNA has been chemically synthesized and used as a primer for DNA polymerase I on phiX-174 plus-strand DNA as template. The sequence of the first 40 nucleotides incorporated onto the decadeoxynucleotide has been determined. This sequence extends further the sequence of the intercistronic region preceding gene G and shows the presence of another termination codon. The sequence was determined by using manganese as the activating cation for DNA polymerase I which allows ribonucleotides to be incorporated as well as deoxyribonucleotides. The ribo-substituted product was then cleaved specifically at the ribonucleotide residues to generate a series of overlapping ribo-terminated fragments whose sequences were sufficient to determine the complete sequence of the first 40 nucleotides. No evidence for misincorporation by DNA polymerase I in the presence of manganese was detected. (+info)
Kinetic analysis of Escherichia coli deoxyribonucleic acid polymerase I.
(61/803)
The kinetic properties of Escherichia coli DNA polymerase I were simplified to those of a 1 deoxynucleotide substrate reaction by the use of polynucleotide templates. With poly(dA)-oligo(dT) as the template-primer complex, Mg2+ decreases the Km of the substrate dTTP but has little or no effect on the Km of the substrate Mg-dTTP, suggesting that multiple pathways involving the binding of Mg2+, dTTP, and Mg-dTTP are operative in forming the active complex. The Km of free Mg2+, extrapolated to zero concentration of substrate (830 = 62 muM), agrees within a factor of 2 with the dissociation constant of magnesium from 4 +/- 1 sites on the enzyme determined previously by binding studies (Slater, J.P., Tamir, I., Loeb, L.A., and Mildvan, A.S. (1972) J. Biol. Chem. 247, 6784-6794). The maximal turnover number with poly(dA) as template is 5.7 +/- 0.7 s-1. Changing the nature of the base in the polydeoxynucleotide template alters the maximal rate of polydeoxynucleotide synthesis by an overall factor of 31 with poly(dC) is greater than poly(dT) is greater than poly(dA) is greater than poly(dG), indicating that pyrimidine templates are copied faster than purine templates. Changing the sugar structure from poly(dA) to poly(rA) causes a 3-fold increase in the rate of template copying. A study of the kinetic effects of all noncomplementary deoxynucleotides with all deoxynucleotide templates, as well as with poly(rA)-oligo(dT), yields complex patterns of activation and inhibition requiring from 1 to 2 additional binding sites for the noncomplementary nucleotides. The kinetically determined affinities of the active site of the enzyme-template-primer complex for the complementary free nucleotide (as measured by Km) generally exceed those for the noncomplementary neuclotides (as measured by KI slope) by 1 or more than 3 orders of magnitude. (+info)
Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis.
(62/803)
The 2,3,6-trideoxysugar l-epivancosamine is the terminal sugar added to the aglycone scaffold in chloroeremomycin, a member of the vancomycin family of glycopeptide antibiotics. Five proteins from the chloroeremomycin biosynthetic cluster, ORF14 and ORF23 to ORF26, have been expressed heterologously in Escherichia coli and purified to near homogeneity, and each has been characterized for an enzymatic activity. These five enzymes reconstitute the complete biosynthesis of TDP-l-epivancosamine from TDP-4-keto-6-deoxy-d-glucose. This process involves C-2 deoxygenation, C-3 amination and methylation, C-5 epimerization, and C-4 ketoreduction. Intermediates and the final product of this pathway have been identified by mass spectrometry and NMR. The pathway established here represents the complete in vitro reconstitution of an unusual sugar for an important class of antibiotics and sets the groundwork for future combinatorial biosynthesis for new bioactive compounds. (+info)
Simultaneous quantitation of intracellular zidovudine and lamivudine triphosphates in human immunodeficiency virus-infected individuals.
(63/803)
Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART. (+info)
Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP.
(64/803)
Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP. (+info)