DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang.
(33/803)
The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5'-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase-based strategies for DNA cloning. Here we characterize a pathway of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3' A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3' G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3' A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3' overhangs. (+info)
Selective interaction of the human immunodeficiency virus type 1 reverse transcriptase nonnucleoside inhibitor efavirenz and its thio-substituted analog with different enzyme-substrate complexes.
(34/803)
Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into the forefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive one is efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In the present study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms of human immunodeficiency virus type 1 RT as well as the association and dissociation rates have been determined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extended to the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both compounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) and as mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in terms of the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showed increasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with lower affinity) binary RT-template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP) complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well below the diffusion limit (on the order of 10(4) M(-1) s(-1)); however, both inhibitors, when bound to the ternary RT-TP-dNTP complex, showed very low dissociation rates, on the order of 10(-4) s(-1) for both compounds, typical of tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiar in their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex. Efavirenz is the first clinically approved NNRTI to show this property. (+info)
The efficiency and fidelity of translesion synthesis past cisplatin and oxaliplatin GpG adducts by human DNA polymerase beta.
(35/803)
DNA polymerase beta (pol beta) is the only mammalian DNA polymerase identified to date that can catalyze extensive bypass of platinum-DNA adducts in vitro. Previous studies suggest that DNA synthesis by pol beta is distributive on primed single-stranded DNA and processive on gapped DNA. The data presented in this paper provide an analysis of translesion synthesis past cisplatin- and oxaliplatin-DNA adducts by pol beta functioning in both distributive and processive modes using primer extension and steady-state kinetic experiments. Translesion synthesis past Pt-DNA adducts was greater with gapped DNA templates than with single-stranded DNA templates. In the processive mode pol beta did not discriminate between cisplatin and oxaliplatin adducts, while in the distributive mode it displayed about 2-fold increased ability for translesion synthesis past oxaliplatin compared with cisplatin adducts. The differentiation between cisplatin and oxaliplatin adducts resulted from a K(m)-mediated increase in the efficiency of dCTP incorporation across from the 3'-G of oxaliplatin-GG adducts. Rates of misincorporation across platinated guanines determined by the steady-state kinetic assay were higher in reactions with primed single-stranded templates than with gapped DNA and a slight increase in the misincorporation of dTTP across from the 3'-G was found for oxaliplatin compared with cisplatin adducts. (+info)
Enhanced antitumor activity of 5-fluorouracil in combination with 2'-deoxyinosine in human colorectal cell lines and human colon tumor xenografts.
(36/803)
We investigated the effects of 2'-deoxyinosine (d-Ino), a modulator yielding thymidine phosphorylase activity, on cellular pharmacology of 5-fluorouracil (FUra) in various human colorectal cell lines and its antitumoral activity when combined with FUra in human xenografts. Associating d-Ino with FUra increased by 38 up to 700 times the sensitivity of HT29 and FUra-resistant SW620 lines, respectively, but not of CaCO2 cells, although high levels of intracellular FdUMP and subsequent higher thymidylate synthase inhibition were observed. Cell death studies confirmed the ability of d-Ino to enhance both early and late apoptosis induced by FUra in HT29 and SW620 but not in CaCo2. Similarly, we showed that associating d-Ino increased by 68 up to 101% the Fas overexpression induced by FUra in HT29 and SW620 but not in CaCo2 cells. Anti-Fas and anti-FasL antibodies both partly reversed this increase of cell sensitivity, thus confirming the role Fas plays in the modulation of FUra toxicity by d-Ino. This Fas component could explain the discrepancy between the lines because CaCO2 has been described as insensitive to Fas-mediated apoptosis. Antitumor activity of the combination was next investigated in nude mice transplanted with SW620. Results showed that although FUra alone has little effect on SW620 xenografts (P > 0.05), associating d-Ino significantly reduced the tumor growth by 57% (P < 0.05). This study suggests that it is possible to reduce both in vitro and in vivo resistance to FUra by modulating the way the drug is converted after cellular uptake. (+info)
The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase.
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The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate. (+info)
Inhibitory effects of of L-2'-deoxyguanosine 5'-triphosphate (L-dGTP) and L-2'-deoxythymidine 5'-triphosphate (L-dTTP) on human telomerase.
(38/803)
Telomerase, which synthesizes telomeric DNA in eukaryotic cells, is classified as a reverse transcriptase. To clarify the recognition of 2'-deoxyribonucleoside 5'-triphosphate (dNTP) chirality by telomerase, we studied the inhibitory effects of L-dGTP on HeLa cell telomerase activity using a quantitative 'stretch PCR' assay. L-dGTP had a weakly inhibitory effect (IC50 = 200 microM) in the presence of 10 microM dGTP. This effect was less obvious when the concentration of dGTP was higher. L-dTTP had a similar inhibitory effect. These findings suggest that telomerase may bind to L-dGTP and L-dTTP, and that the ability of telomerase to bind to dGTP or dTTP is changed. (+info)
Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP.
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Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3, 5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer beta-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs. (+info)
Pharmacokinetics of zidovudine phosphorylation in human immunodeficiency virus-positive thai patients and healthy volunteers.
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We assessed the pharmacokinetics of zidovudine (ZDV) in plasma and intracellular ZDV phosphate anabolites in peripheral blood mononuclear cells in Thai human immunodeficiency virus (HIV) type 1-infected patients and healthy volunteers. The plasma ZDV area under the concentration-time curve from 0 to 6 h (AUC(0-6)) was similar in patients and healthy volunteers (32.77 and 22.77 micromol/liter. h, respectively; confidence interval, -3.37 to 19. 92). Although the concentration of ZDV triphosphate (ZDVTP) was similar in the two groups, the ZDV monophosphate (ZDVMP) AUC(0-6) was significantly greater in HIV patients (1.12 pmol/10(6) cells) than in healthy volunteers (0.15 pmol/10(6) cells). In agreement with previously published data obtained with Caucasians, the significant difference in intracellular phosphorylation in Thai volunteers and HIV patients is primarily due to ZDVMP. Comparing the data from this study with the data obtained with Caucasians suggests no marked ethnic differences in ZDV phosphorylation. (+info)