No abstract

DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. It requires two divalent cations, forming an active E-M2+-NTP-M2+ complex. The solution structure of the free enzyme consists of a five-stranded mixed beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-helix II [Abeygunawardana, C., et al. (1995) Biochemistry 34, 14997-15005]. Assignments of backbone 15N and NH resonances and side chain 15N and NH2 resonances of the quaternary complex were made by 1H-15N HSQC titrations of the free enzyme with MgCl2 followed by equimolar AMPCPP/MgCl2. H(alpha) assignments were made by 1H-15N 3D TOCSY HSQC, and 1H-13C CT-HSQC spectra and backbone and side chain 1H and 13C assignments were made by 3D HCCH TOCSY experiments. Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co2+ and Mn2+ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38. The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 phi restraints for the protein, 11 intramolecular NOEs for bound Mg2+ AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co2+ to the three phosphorus atoms of Co3+(NH3)4AMPCPP from paramagnetic effects of Co2+ on their T1 values. The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites. The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on beta-strand A and with Ile-80 on beta-strand D. The 6-NH2 group of adenine approaches the side chain NH2 of Asn-119. This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group. The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I. The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates.

Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite.The Nudix hydrolases comprise a large family of proteins characterized by the highly conserved array of amino acids GX 5 EX 7 REUXEEXGU, where U represents a bulky, hydrophobic, amino acid, usually Ile, Leu, or Val (1). A recent BLAST (2) search of the sequence data banks has revealed more than 300 putative proteins from over 80 species containing this amino acid motif, the Nudix box ( Fig. 1). We have been systematically identifying and characterizing the enzymatic activities associated with these proteins, and we have found that almost all of the major substrates for these enzymes are nucleoside diphosphates linked to some other moiety, x, hence the acronym "Nudix." The range of substrates acted on by various members of the family includes ribo-and deoxyribonucleoside triphosphates, nucleotide sugars, dinucleoside polyphosphates, NADH, and ADP-ribose. These substances are potentially toxic to the cell, signaling molecules, or metabolic intermediates whose concentrations require modulation during changes in the cell cycle or during periods of stress. We have suggested that the role of the Nudix hydrolases is to sanitize or modulate the accumulation of these metabolites (1). Since the Nudix box is common to all of these enzymes, their specificity for the individual substrates must lie somewhere distal to the conserved region. In this paper, we describe the cloning and characterization of four ADP-ribose pyrophosphatases, and we identify a proline residue downstream of the conserved sequence common to members of this subfamily of Nudix hydrolases. Furthermore, we have observed that other recurring amino acids in this same region are predictive of two other subfamilies of the Nudix hydrolases, the dinucleoside polyphosphate pyrophosphatases and the NADH pyrophosphatases.We also demonstrate that ADP-ribose pyrophosphatase activity may play a role in tellurite resistance, since overexpression o...

The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3 -5 helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.The epidemic caused by infection by the hepatitis C virus (HCV) 1 is still a global crisis despite recent therapeutic advancements (1). Because HCV cannot be conventionally cultivated in cell culture and the only other host is the chimpanzee, the enzymes encoded by HCV have been studied intensely as targets for rational drug design. One key viral enzyme is the multifunctional nonstructural protein 3 (NS3), which possesses a serine protease activity and an ATPase function that fuels the ability of the protein to unwind RNA and DNA duplexes. Although it is clear that both the protease and helicase functions are necessary for viral replication (2), it is not clear whether the two functions, which reside in independent protein domains, cooperate in any manner (3).To examine possible effects of the NS3 protease on its helicase function, the activities of the full-length NS3 protein were rigorously compared with the same protein lacking the protease and also with recombinant proteins in which the protease is replaced with other non-HCV peptides. The experiments were designed to uncover effects of the NS3 protease domain on its helicase function which are either specific or nonspecific. Nonspecific effects are defined as those that can be duplicated by peptides not derived from HCV NS3, whereas specific effects cannot.All recombinant proteins used in this study ( Fig. 1) were derived from the same infectious clone of HCV genotype 1a (4). NS3 spans amino acids 1027-1659 of the ϳ3,000-amino acid long polyprotein encoded by HCV. At the N terminus resides the pro...

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of ␤-D-2-deoxy-2-fluoro-2-C-methyluridine-5-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ؎ 0.050 M (mean ؎ standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 M). Crossresistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.Hepatitis C virus (HCV) currently affects more than 170 million people worldwide. Approximately 70% of infected individuals develop chronic hepatitis, among whom about 20% will develop liver cirrhosis and fibrosis and up to 5% will progress to hepatocellular carcinoma (2). The current standard of care (SOC), which combines pegylated alpha interferon (PegIFN-␣) and ribavirin (RBV), has limited efficacy in providing a sustained virological response (SVR), especially in individuals with HCV genotype 1 (ϳ50%), the most prevalent genotype in Western countries (8,11,35). The impact of genetic diversity of HCV in patients receiving SOC therapy has been reviewed (26): SVR rates are higher in patients infected with genotype 2 or 3 (ϳ80%), patients infected with genotype 4 appear to have a slightly better SVR rate (ϳ60%) than patients infected with genotype 1, and patients infected with genotypes 5 and 6 may achieve an SVR at a level between those of genotypes 1 and 2/3. In addition to the variability in efficacy, the lengthy treatment (24 to 48 w...

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked P-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed P-sheet connected by the loop I-a-helix I-loop I1 motif, by two tight tums, and by loop 111, and terminated by loop IV-a-helix I1 (4) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure ofMutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the P-sheet sandwiched between the two a-helices, forming an a f , ! ? fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-55861. The tertiary structure of MutT shows these residues to be near each other along the loop I-helix I region of the enzyme. A cluster of five glutamate residues (41,53, 56,57, and 98) form a patch of strongly negative electrostatic potential likely constituting the metal binding site. This site is contiguous with a deep cleft between P-strands A, C, and D and loop I which may contribute to the nucleotide binding site. This location of the active site is consistent with mutagenesis studies and with sequence homologies among MutT-like pyrophosphohydrolases.The MutT enzyme, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked ,!?-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al., 1991;Weber et al., 1992). Like other enzymes which catalyze substitution at the electron-rich ,!?-phosphorus, this enzyme requires two divalent cations for activity (Frick et al., 1994). The biological role of this enzyme is to prevent ' This work was supported in part by National Institutes of Health Grants DK28616 (to A.S.M.), GM18649 (to M.J.B.), and GM36358 (which supports A.G.G.).A complete listing of the distance restraints derived from the NOE data has been deposited at the Brookhaven Protein Data Bank (Chemistry Department, Brookhaven National Laboratory, Upton, NJ) together with the atomic coordinates of the family of 15 acceptable structures (file name IMUT).