Inhibition of HIV-1 reverse transcriptase-catalyzed DNA strand transfer reactions by 4-chlorophenylhydrazone of mesoxalic acid.

DNA strand transfer reactions occur twice during retroviral reverse transcription catalyzed by HIV-1 reverse transcriptase. The 4-chlorophenylhydrazone of mesoxalic acid (CPHM) was found to be an inhibitor of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Using a model strand transfer assay system described previously [Davis, W. R., et al. (1998) Biochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM was found to target the RNase H activity of HIV-1 reverse transcriptase. DNA polymerase activity was not significantly affected by CPHM; however, it did inhibit the polymerase-independent RNase H activity with an IC(50) of 2.2 microM. In the absence of DNA synthesis, CPHM appears to interfere with the translocation, or repositioning, of RT on the RNA.DNA template duplex, a step required for efficient RNA hydrolysis by RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase; little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV or AMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional 4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity, suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid in inhibitor function, Mg(2+) was found to chelate directly to CPHM with a K(d) of 2.4 mM. Together, these studies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.

Asymmetry in the autophosphorylation of the two-component regulatory system transmitter protein nitrogen regulator II of Escherichia coli.

Autophosphorylation of the homodimeric two-component system transmitter protein nitrogen regulator II (NRII; also NtrB) of Escherichia coli is the first step in the activation of nitrogen-regulated (Ntr) gene transcription. We show that the autophosphorylation of NRII was asymmetric, with phosphorylation of the first and second subunits of the dimer displaying different equilibria (under our experimental conditions K(1) approximately 0. 345, K(2) approximately 0.0044). Phosphorylation of both subunits of NRII was rapid, but the very rapid reversal of the phosphorylation of the second subunit was responsible for the equilibrium position of the reaction. Complete phosphorylation of NRII was only observed under conditions where ADP, a product of the autophosphorylation reaction, was removed by an enzymatic system. Purified, doubly phosphorylated NRII (NRII approximately P(2)) was stable in the absence of nucleotides at 0 degrees C but was dephosphorylated to the hemiphosphorylated form at 37 degrees C. In the presence of a low concentration of ADP, half of the phosphoryl groups from NRII approximately P(2) were rapidly dephosphorylated, while the remaining phosphoryl groups were slowly dephosphorylated. Experiments with heterodimers containing wild-type and mutant, nonphosphorylatable subunits suggested that the asymmetry of NRII autophosphorylation was not preexisting but resulted from the autophosphorylation of one subunit.

Inhibitors of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

The discovery and characterization of new inhibitors of HIV-1 reverse transcriptase (RT) is an important step toward understanding the mechanism of this multifunctional polymerase. We describe the identification of novel inhibitors of HIV-1 RT-catalyzed reactions utilizing a nucleic acid model system designed to mimic the essential features of DNA strand transfer reactions catalyzed by HIV-1 RT. This reaction requires the DNA polymerase and RNase H activities of RT, as well as the translocation of DNA from one template strand to another. In addition to the discovery of new inhibitors of DNA polymerase activity, two classes of inhibitors were identified that inhibit different steps of the DNA strand transfer reaction. One class of these, exemplified by actinomycin D, inhibits DNA strand transfer by interfering with the transfer of the DNA intermediate onto the acceptor template. The second class of strand transfer inhibitor, exemplified by the chlorophenylhydrazone of mesoxalic acid, was found to inhibit the ribonuclease H (RNase H) activity of HIV-1 RT under strand transfer conditions. This inhibitor is a potent and specific inhibitor of RNase H activity, which displays no inhibition of either DNA-dependent or RNA-dependent DNA polymerase activity. Together, these three inhibitors block different steps reverse transcription and will be valuable in studying the mechanism of multistep reactions such as DNA strand transfer. In addition, these new inhibitors of in vitro reverse transcription point to new strategies for the intervention of retroviral DNA replication and could be useful in the development of new HIV-1 therapeutic strategies.

Actinomycin D inhibition of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein.

Actinomycin D was found to be a potent inhibitor of HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions. Using an oligonucleotide model system, actinomycin D inhibition of DNA strand transfer was examined to elucidate the mechanism of inhibition and further define the mechanism of DNA strand transfer. Our results show that actinomycin D inhibits HIV-1 reverse transcriptase catalyzed DNA strand transfer without inhibiting RNA-dependent or DNA-dependent DNA polymerase activity. Actinomycin D was found to strongly inhibit annealing of a primary DNA product to the DNA acceptor template, preventing the formation of a key reaction intermediate. The HIV-1 nucleocapsid protein has been shown to participate in catalytic events during reverse transcription including DNA strand transfer. Recombinant nucleocapsid protein was used in conjunction with actinomycin D in this model system to investigate how NC may participate in the mechanism of inhibition by actinomycin D and in DNA strand transfer. The inclusion of nucleocapsid protein was found to partially relieve both DNA annealing and strand transfer inhibition caused by actinomycin D. This study suggests a potential new mechanism for inhibiting retroviral replication by preventing the formation of replication intermediates.

Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein.

The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia coli plays an important role in the regulation of nitrogen assimilation by controlling the uridylylation state of the PII signal transduction protein (PII) in response to intracellular signals. The reversible uridylylation of PII indirectly controls the activity of PII receptors that regulate transcription from nitrogen-regulated promoters and the activity of glutamine synthetase. Here, we present a detailed analysis of the uridylyltransferase and uridylyl-removing activities and their regulation by the small molecule effectors ATP, 2-ketoglutarate, and glutamine. Several important features of enzyme mechanism and regulation were elucidated. Mg2+ appeared to be the physiologically relevant metal ion cofactor for both transferase and uridylyl-removing activities. The transferase reaction proceeded by an ordered bi-bi kinetic mechanism, with PII binding before UTP and pyrophosphate (PPi) released before PII-UMP. The uridylyl-removing reaction proceeded with rapid equilibrium binding of substrate and random release of products. Both reactions were activated by ATP and 2-ketoglutarate, which did so by binding only to PII and PII-UMP. The binding of these effectors to PII and PII-UMP was characterized. Glutamine inhibited the transferase reaction by inhibiting the chemistry step, while glutamine provided nonessential mixed-type activation of the uridylyl-removing activity, lowering the apparent Km and increasing kcat. Our data were consistent with the hypothesis that all effects of glutamine are due to the binding of central complexes at a single glutamine site. By comparing the effects of the activators with their reported in vivo concentrations, we conclude that in intact cells the uridylylation state of PII is regulated mainly by the glutamine concentration and is largely independent of the 2-ketoglutarate concentration. Our kinetic data were consistent with the hypothesis that both transferase and uridylyl-removal reactions occurred at a single active center on the enzyme.

The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed.

Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli.

Nitrogen-regulation of gene transcription in Escherichia coli results from the regulation of the phosphorylation state of the enhancer-binding transcription factor NRI (NtrC). We examined the regulation of NRI phosphorylation in a reconstituted bicyclic cascade system containing four regulatory proteins: NRI, the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate the signal transduction protein PII, and the kinase/phosphatase NRII (NtrB), which is a PII receptor that phosphorylates and dephosphorylates NRI. In this reconstituted system, the phosphorylation state of NRI was regulated reciprocally by the small molecule effectors glutamine, which prevented the accumulation of NRI-P, and 2-ketoglutarate, which caused accumulation of NRI-P. Regulation of the bicyclic system by glutamine was exclusively due to sensation and signal-transduction by the UTase/UR-PII monocycle, which was observed to function essentially as a glutamine-sensing apparatus. In contrast, regulation of NRI phosphorylation by 2-ketoglutarate, which binds to PII, was due to direct regulation of the NRII-PII interaction and the rate of NRI-P dephosphorylation. Thus, the PII protein transduces the glutamine signal to the NRII-NRI monocycle in the form of its uridylylation state and is also the receptor of the antagonistic 2-ketoglutarate signal, which blocks the activity of unmodified PII.

Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment.

Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S). Design of the template-primer allowed the probe's position within the DNA-protein complex to be varied by stepwise extension of the primer strand upon addition of the appropriate deoxynucleoside triphosphates (dNTP). The TMR acceptor probe occupied seven different positions in the template-primers, five in the single stranded region and two in the double stranded region. The efficiency of energy transfer was determined at each position by calculating the integrated area of the fluorescein emission peak in the presence and absence of acceptor. Results indicate that the FRET efficiency varied in a sinusoidal fashion with a periodicity of approximately 10 base pairs and that the data could be fitted to an equation derived from a simple model formulated on the basis of helical structure. The data support the conclusion that the single stranded template portion of a DNA template-primer adopts a helical conformation when bound to the KF. The results of this study further support FRET as a useful method for the determination of structure and conformation in protein-DNA complexes.

Recombinant HIV-1 nucleocapsid protein accelerates HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions and modulates RNase H activity.

The effect of recombinant nucleocapsid protein (NCp7) from human immunodeficiency virus type 1 (HIV-1) on HIV-1 reverse transcriptase (HIV-1 RT) catalyzed DNA strand transfer reactions has been studied using kinetic methods with a defined template--primer model system. NCp7 is shown to modulate both the rate and the efficiency of DNA strand transfer synthesis. Evidence is presented that supports the role of NCp7 in catalyzing the annealing of a nascent DNA intermediate and RNA acceptor template during strand transfer. NCp7 was also found to enhance the ribonuclease H activity of HIV-1 RT and change the specificity of RNA hydrolysis, suggesting a direct role of NCp7 in HIV-1 RT catalyzed strand transfer. The implications of these findings for retroviral reverse transcription are addressed.

Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

The fidelity of DNA strand transfer reactions catalyzed by human immunodeficiency virus type 1 reverse transcriptase has been studied in vitro. A model system involving two sequential DNA strand transfers was developed to simulate the process of forced copy-choice recombination. A propensity for nucleotide misincorporation at the junction of the strand transfer, as determined by DNA sequencing of the reaction products, was found consistent with a model involving the addition of nontemplate-directed nucleotides prior to the transfer of nascent DNA onto the accepting RNA template [Peliska, J. A., & Benkovic, S. J. (1992) Science 258, 1112]. The kinetic and mechanistic factors that may dictate which nucleotide bases are incorporated at recombination sites during strand transfer and the possible consequences of recombination-induced mutagenesis in vivo are discussed.

Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship between the polymerase and RNase H activities.

The spatial and temporal relationship between the polymerase and RNase H activities of human immunodeficiency virus type 1 reverse transcriptase has been examined by using a 40-mer RNA template and a series of DNA primers of lengths ranging from 15 to 40 nucleotides, hybridized to the RNA, as substrates. The experiments were executed in the absence and presence of heparin, an efficient trap to sequester any free or dissociated reverse transcriptase, thus facilitating the study of events associated with a single turnover of the enzyme. The results indicate a spatial separation of 18 or 19 nucleotides between the two sites. To examine the effect of concomitant polymerization on the RNase H activity, the substrate was doubly 5' end labeled on the RNA and DNA. This enabled the study of RNase H activity as a function of polymerization in a single experiment, and the results in the absence and presence of heparin indicate a tight temporal coupling between the two activities.

Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

Two DNA strand transfer reactions occur during retroviral reverse transcription. The mechanism of the first, minus strand strong-stop DNA, transfer has been studied in vitro with human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) and a model template-primer system derived from the HIV-1 genome. The results reveal that HIV-1 RT alone can catalyze DNA strand transfer reactions. Two kinetically distinct ribonuclease (RNase) H activities associated with HIV-1 RT are required for removal of RNA fragments annealed to the nascent DNA strand. Examination of the binding of DNA.RNA duplex and single-stranded RNA to HIV-1 RT during strand transfer supports a model where the enzyme accommodates both the acceptor RNA template and the nascent DNA strand before the transfer event is completed. The polymerase activity incorporated additional bases beyond the 5' end of the RNA template, resulting in a base misincorporation upon DNA strand transfer. Such a process occurring in vivo during retroviral homologous recombination could contribute to the hypermutability of the HIV-1 genome.

Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4.

The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

Sulfuryl transfer catalyzed by phosphokinases.

Adenosine 5'-sulfatopyrophosphate is a substrate for nucleoside diphosphate kinase. The reaction appears to proceed through a ping-pong mechanism analogous to the physiological reaction involving ATP, presumably by way of a sulfohistidine intermediate. Unlike the phosphoryl transfer reactions, the corresponding sulfuryl transfers catalyzed by nucleoside diphosphate kinase do not have a strict divalent metal requirement. The estimated rate constants for the metal- and nonmetal-catalyzed sulfuryl transfers differ by less than an order of magnitude and are approximately 1000-fold slower than the corresponding phosphate transfers. These results suggest that the role of the metal ion in nucleoside diphosphate kinase is to coordinate the alpha, beta-phosphates of the substrate. Sulfuryl and phosphoryl transfer probably occur through dissociative transition states.

Synthesis and study of (Z)-3-chlorophosphoenolpyruvate.

(Z)-3-Chlorophosphoenolpyruvate has been synthesized by the reaction of 3,3-dichloropyruvic acid with trimethylphosphite, followed by deesterification. This compound is a competitive inhibitor of pyruvate kinase and phosphoenolpyruvate carboxylase. Pyruvate kinase is not inactivated upon prolonged incubation with the compound, but phosphoenolpyruvate carboxylase is slowly inactivated (t1/2 = 5 h). The compound is a substrate for both enzymes, being acted upon by pyruvate kinase approximately 0.1% as rapidly as phosphoenolpyruvate itself. In the case of phosphoenolpyruvate carboxylase, the compound is converted into a 3:1 mixture of chloropyruvate and chlorooxalacetate, at an overall rate that is about 25% the carboxylation rate for phosphoenolpyruvate.

Sulfuryl transfer catalyzed by pyruvate kinase.

Sulfoenolpyruvate, the analogue of phosphoenolpyruvate in which the phosphate ester has been replaced by a sulfate ester, has been synthesized in three chemical steps from ethyl bromopyruvate in 40% overall yield. This compound is a substrate for pyruvate kinase, producing pyruvate and adenosine 5'-sulfatopyrophosphate. The latter compound has been identified by NMR spectroscopy and by comparison with an authentic sample. Sulfuryl transfer from sulfoenolpyruvate is 250-600-fold slower than phosphate transfer from phosphoenolpyruvate under identical conditions. Sulfoenolpyruvate is not a substrate for phosphoenolpyruvate carboxylase. Kinetic studies reveal that it does not bind to the active site; instead, it binds to the site normally occupied by glucose 6-phosphate and activates the enzyme in a manner similar to that shown by glucose 6-phosphate.