Binding Of dATP Research Articles

NetSci: A Library for High Performance Biomolecular Simulation Network Analysis Computation.

We present the NetSci program-an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2'-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

Structural Investigation of Allosteric Regulation in Class III Ribonucleotide Reductases

Ribonucleotide reductases (RNRs) are essential enzymes that use radical‐based chemistry to catalyze the reduction of ribonucleotides to deoxyribonucleotides. Each RNR class uses a different cofactor to generate a catalytically‐essential thiyl radical species in the enzyme active site. Anaerobic (class III) RNRs employ an oxygen‐sensitive glycyl radical cofactor installed within the glycyl radical domain (GRD) that is adjacent to the active site, whereas aerobic (class Ia) RNRs employ long‐range radical transfer between the catalytic subunit and a di‐iron/tyrosyl radical cofactor housed in the b2 subunit. To maintain intracellular nucleotide pool balance, RNRs are allosterically regulated. For the prototypical class Ia RNRs from Escherichia coli, this allosteric activity regulation involves dATP binding to the N‐terminus of the catalytic subunit in a region known as the ‘cone domain’, resulting in an association between the cone domain and the b2 subunit that prevents radical transfer between subunits. Despite not requiring a b2 subunit, some class III RNRs (e.g. Streptococcus thermophilus)have a cone domain and are allosterically inhibited by dATP. In this study, we investigate allosteric activity regulation for such a class III RNR, the class III RNR from Streptococcus thermophilus(StNrdD). We use a newly developed LC‐MS/MS based activity assay to show that dATP downregulates StNrdD activity and ATP upregulates activity as is true for class Ia RNRs. Using crystallography, we obtain structural data showing that ATP and dATP both bind to the cone domain, and that both the cone domain and GRD are flexible. In particular, in one monomer of StNrdD dimer, the cone domain contacts the GRD, and both domains are ordered, and in the other monomer, the cone domain is positioned away from the GRD and the GRD is disordered. These observations led us to hypothesize that the cone domains of class III RNRs, like those of class Ia RNR, regulate activity through promotion or inhibition of the radical transfer step, but instead of controlling the position of b2, the position of the GRD is allosterically regulated. In this presentation, we show the data described above and show recent cryogenic electron microscopy of ATP‐bound and dATP‐bound states of StNrdD, which reveals differential positioning of the cone domains in the presence of these allosteric effectors. We also present our current thinking about the mechanism of allosteric regulation of activity in a class III RNR.

Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy.

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary α2β2 complex between the catalytic (α2) and radical generating (β2) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the β subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in β results in formation of an unusual β4 tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the α4β4 octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical α2β2 complex.

A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

Binding studies of a putative C. pseudotuberculosis target protein from Vitamin B12 Metabolism

Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis’s Vitamin B12 metabolism and related pathways to reduce the pathogen’s virulence in its hosts.

Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a β2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2β2 state allowing for radical transfer from β2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4β4 ring-like state. Here, we investigate whether the α4β4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α2, S39R-α2, S39F-α2, E42K-α2, or L43Q-α2) that disrupt the α4β4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4β4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-β interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-β interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α2 or E42K-α2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.

Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592.

The sterile alpha motif (SAM) and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) constitute a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site 1 (AS1), induces formation of the tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic sites for all possible combinations of dNTP binding at both sites. Here, we show that the apparent Km values of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP < dTTP. Interestingly, dCTP binding at AS2 significantly reduces the dCTP hydrolysis rate, which is restored to a rate comparable to that of other dNTPs upon dGTP, dATP, or dTTP binding at AS2. Strikingly, a phosphomimetic mutant, Thr592Asp SAMHD1 as well as phospho-Thr592, show a significantly altered substrate specificity, with the rate of dCTP hydrolysis being selectively reduced regardless of which dNTP binds at AS2. Furthermore, cyclin A2 binding at the C-terminus of SAMHD1 induces the disassembly of the SAMHD1 tetramer, suggesting an additional layer of SAMHD1 activity modulation by cyclin A2/CDK2 kinase. Together, our results reveal multiple allosteric mechanisms for controlling the rate of dNTP destruction by SAMHD1.

Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.DOI: http://dx.doi.org/10.7554/eLife.07141.001

Flavonoid‐Based Organometallics with Different Metal Centers – Investigations of the Effects on Reactivity and Cytotoxicity

AbstractFlavonol‐derived OsII(cym) and RhIII‐Cp* complexes were synthesized and the impact of the metal center on aqueous stability, reactivity toward biomolecules, and cytotoxic activity was compared to RuII analogs. The RhIII complexes were the most stable to ligand release in aqueous solution and showed the highest preference for ubiquitin binding. Investigations on the reactivity toward nucleoside triphosphates revealed the clear affinity of RhIII to 5′‐dATP, whereas RuII and OsII prefer binding to 5′‐dGTP. Simultaneous incubation with amino acids and nucleoside triphosphates reveals the preference toward amino acids, indicating that binding to proteins might be a key step in the mechanism of action of this compound class. The complexes exhibit in vitro anticancer activities in the high nM to low μM range, confirming the flavonol scaffold as a promising O,O‐chelating ligand system for the development of anticancer active organometallics.

Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Relative Binding Free Energies of Adenine and Guanine to Damaged and Undamaged DNA in Human DNA Polymeraseη: Clues for Fidelity and Overall Efficiency.

Human DNA polymerase η (Pol η) plays an essential protective role against skin cancer caused by cyclobutane thymine–thymine dimers (TTDs), a frequent form of DNA damage arising from exposure to the sun. This enzyme rescues stalled replication forks at the TTDs by inserting bases opposite these DNA defects. Herein we calculate binding free energies for a free deoxyribose nucleotide triphosphate, dATP or dGTP, to Pol η complexed with undamaged or damaged DNA. The calculations indicate that the binding of dATP to the enzyme–DNA complex is thermodynamically favored for TTD-containing DNA over undamaged DNA, most likely because of more extensive hydrogen-bonding interactions between the TTD and the enzyme that hold the TTD more rigidly in place. The calculations also illustrate that dATP binding is thermodynamically favored over dGTP binding at both thymine positions of the TTD, most likely due to more persistent and stable hydrogen-bonding interactions between the TTD and dATP than between the TTD and dGTP. This free energy difference is slightly greater for binding at the 5′ thymine position than at the 3′ thymine position, presumably because of stabilization arising from the A:T base pair formed at the 3′ position of the TTD in the previous step of Pol η function. All of these trends in binding free energies are consistent with experimental measurements of binding strength, fidelity, processivity, and overall efficiency. The insights gained from this analysis have implications for drug design efforts aimed at modifying the binding properties of this enzyme for improving cancer chemotherapy treatments.

IRBIT promotes allosteric inhibition of ribonucleotide reductase.

IRBIT inhibits ribonucleotide reductase (RNR) by stabilizing dATP binding to the RNR activity site.

Uncoupling of Allosteric and Oligomeric Regulation in a Functional Hybrid Enzyme Constructed from Escherichia coli and Human Ribonucleotide Reductase

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

2-deoxy-ATP Alters Myosin Structure to Enhance Cross-Bridge Cycling and Improve Cardiac Function

We previously reported that increased cellular 2-deoxy-ATP (dATP) augments contraction, enhancing the rate and magnitude of both contraction and relaxation in intact cardiomyocytes, force production in skinned cardiac muscle, and fractional shortening of the whole heart. To better understand the mechanism by which dATP enhances cardiac function, we used molecular dynamics (MD) simulations to study the pre- and post-powerstroke states of myosin (PDB ID: 1VOM and 1MMA) bound to either Mg2+.(d)ADP.Pi (pre-powerstroke) or Mg2+.(d)ADP (post-powerstroke) for 50ns. In both states, dATP binding to myosin alters the conformation of the nucleotide binding pocket and the actin binding surface. In the pre-powerstroke state, myosin made fewer contacts with dADP than with ADP and this structural change translated to increased exposure of polar residues on the actin-binding surface of myosin. Since the initial acto-myosin interaction is primarily electrostatic, the affinity of myosin for actin may thus be enhanced with dADP.Pi. In post-powerstroke simulations, dADP binding reduced the exposure of actin binding residues on myosin somewhat. This may translate into improved ability for myosin detachment from actin at the end of the powerstroke in the presence of dADP vs. ADP. Furthermore, stopped-flow spectroscopy demonstrated that myosin has 40-80% weaker binding affinity for dADP than ADP, which may also contribute to faster myosin detachment. Together, these data demonstrate that dADP binding to myosin significantly alters the conformation of myosin, which may translate to faster cross-bridge cycling due to both enhanced myosin binding in the pre-powerstroke state and faster myosin detachment from actin in the post-powerstroke state. Ongoing studies examining the effect of enhanced cross-bridge cycling with elevated dATP in infarcted hearts suggest that dATP may reduce the loss of systolic function. Supported by HL111197 (MR), WT085309 (MAG), GM50789 (VD) and T32EB001650 (SGN).

Mathematical modeling of the formation of apoptosome in intrinsic pathway of apoptosis

Caspase-9 is the protease that mediates the intrinsic pathway of apoptosis, a type of cell death. Activation of caspase-9 is a multi-step process that requires dATP or ATP and involves at least two proteins, cytochrome c and Apaf-1. In this study, we mathematically model caspase-9 activation by using a system of ordinary differential equations (an ODE model) generated by a systems biology tool Simpathica—a simulation and reasoning system, developed to study biological pathways. A rudimentary version of “model checking” based on comparing simulation data with that obtained from a recombinant system of caspase-9 activation, provided several new insights into regulation of this protease. The model predicts that the activation begins with binding of dATP to Apaf-1, which initiates the interaction between Apaf-1 and cytochrome c, thus forming a complex that oligomerizes into an active caspase-9 holoenzyme via a linear binding model with cooperative interaction rather than through network formation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-009-9022-y) contains supplementary material, which is available to authorized users.

Bacillus subtilis SsbA and dATP regulate RecA nucleation onto single-stranded DNA

Bacillus subtilis RecA preferentially hydrolyzes dATP over ATP and supports an efficient DNA strand exchange reaction in the presence of dATP when compared to ATP. Saturating amounts of SsbA, independently of the order of addition, reduce the single-stranded (ss) DNA-dependent dATPase activity of RecA, and block the ATPase activity. SsbA added prior to RecA slightly stimulates the dATP-dependent DNA strand exchange activity, whereas added after RecA greatly enhances the extent of strand exchange. In the presence of ATP, 10 times more RecA is required to achieve a comparable level of strand exchange than in the presence of dATP. We propose that dATP binding and hydrolysis as well as SsbA provide different levels of regulation of the dynamic RecA nucleoprotein filament.

Structures of Heparin-Derived Tetrasaccharide Bound to Cobra Cardiotoxins: Heparin Binding at a Single Protein Site With Diverse Side Chain Interactions

Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid, and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle, we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin-CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.

Kinetic Effect of a Downstream Strand and Its 5′-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase λ

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.