Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+

The use of Mn(2+) as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg(2+) often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn(2+). The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg(2+) and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn(2+), the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn(2+), it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg(2+), highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn(2+) to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg(2+), this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn(2+) may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg(2+).     

Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese

All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg(2+) is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn(2+) in vitro. Here we report on the effects of Mn(2+) and Mg(2+) on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn(2+) (0.05-0.25 mm). Peak activity in the presence of Mg(2+) was observed in the range of 0.1-0.5 mm and was significantly reduced at concentrations >2 mm. Steady-state kinetic analyses revealed that Mn(2+) increases the catalytic activity of pol iota by approximately 30-60,000-fold through a dramatic decrease in the K(m) value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of approximately 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mm Mg(2+), respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mm Mn(2+). Low levels of Mn(2+) also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn(2+) even when Mg(2+) is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn(2+) rather than Mg(2+), as tacitly assumed.     

Bypass of N²-ethylguanine by human DNA polymerase κ

The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N(2)-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3' termini opposite N(2)-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N(2)-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn(2+) compared to Mg(2+). In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N(2)-ethyl-Gua bypass is Mg(2+)-dependent. Strikingly, Mn(2+) activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N(2)-ethyl-Gua and Gua compared to that detected upon Mg(2+) activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K(m) values for dCTP insertion opposite N(2)-ethyl-Gua and Gua with Mn(2+) activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn(2+) compared to Mg(2+) indicating that DNA pol κ preferentially utilizes Mg(2+) activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N(2)-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn(2+) suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.     

DNA polymerase beta: pre-steady-state kinetic analyses of dATP alpha S stereoselectivity and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis

The first pre-steady-state kinetic analysis of the stereoselectivity of a DNA polymerase, Pol beta from rat brain, toward Rp and Sp isomers of dATPalphaS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPalphaS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (k(pol)) and the apparent dissociation constant (K(d,app)) were measured with dATP, Rp-dATPalphaS, and Sp-dATPalphaS in the presence of Mg(2+), Mn(2+), or Cd(2+). The results indicate that wild type (WT) polymerase (Pol) beta can incorporate both Sp- and Rp-dATPalphaS in the presence of Mg(2+), but Sp is the preferred isomer. The stereoselectivity, defined as (k(pol)/K(d))(Sp)/(k(pol)/K(d))(Rp) (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg(2+). When Mg(2+) was substituted with Mn(2+) and Cd(2+), the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol beta complexes, as well as previous steady-state kinetic studies of other DNA polymerases. In addition, the D276R mutant was designed to introduce a potential extra hydrogen bonding interaction between the arginine side chain and the pro-Sp oxygen of the alpha-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPalphaS (Sp/Rp ratio = 1.5, 3.7, and 1.5 for Mg(2+), Mn(2+), and Cd(2+), respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPalphaS relative to WT Pol beta, suggesting that this mutant Pol beta can be used to incorporate Rp-dNTPalphaS into DNA oligomers.     

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as "sym/sub." Formation of 3D(pol)-sym/sub complexes is slow owing to a slow rate of association (0.1 microM(-1) s(-1)) of 3D(pol) and sym/sub and a slow isomerization (0. 076 s(-1)) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s(-1) at 22 degrees C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K(0.5) of 37 microM. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t((1)/(2)) = 2 h at 22 degrees C) and appear to contain only a single polymerase monomer. In the presence of Mg(2+), AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s(-1), respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 degrees C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 degrees C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.     

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol)

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.     

Altered specificity of transfer-ribonucleic acid nucleotidyltransferase in the presence of manganese

1. s-RNA nucleotidyltransferase incorporated CMP into phosphodiesterase-treated s-RNA more rapidly in the presence of Mg(2+) (10mm) than in the presence of Mn(2+) (2mm). UMP was incorporated more rapidly in the presence of Mn(2+), and at high ionic strength the incorporation of CMP was also more rapid in the presence of Mn(2+). 2. The capacity of phosphodiesterase-treated s-RNA for CMP, UMP and AMP was increased in the presence of Mn(2+). Terminal sequences of more than one UMP or AMP residue were synthesized, but these atypical reactions were inhibited when CTP was added. CMP was incorporated rapidly to form -pCpC terminal sequences and then more slowly as longer chains were formed, but very little CMP was incorporated into s-RNA-pCpCpA. 3. CMP was incorporated into phosphodiesterase-treated 5s RNA and ribosomal RNA to form short chains of polyC attached to the primer RNA. This reaction was inhibited by the presence of s-RNA. 4. A small Mn(2+)-dependent incorporation of CMP was also primed by poly(A).(U) and poly(C).(I).     

Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.     

Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron <Emphasis Type="Italic">Sc</Emphasis>.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg(2+) to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis-Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5'-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca(2+), Mn(2+), Ni(2+), Zn(2+), Cd(2+), Pb(2+), and [Co(NH(3))(6)](3+) on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5gamma. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg(2+). For example, the presence of only 5% Ca(2+) relative to Mg(2+) results in a decrease in the maximal turnover rate k (cat) by 50%. Ca(2+) thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg(2+) in the folded state, the latter being indicative of at least one specific Ca(2+) binding pocket interfering directly with catalysis. Similar results are obtained with Mn(2+), Cd(2+), and [Co(NH(3))(6)](3+). Ni(2+) is a much more powerful inhibitor and the presence of either Zn(2+) or Pb(2+) leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg(2+) and raises the question of biological relevance at least in the case of Ca(2+).     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.     

Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg(2+) or Mn(2+) as activating ions. 2. The enzyme assayed with Mg(2+) and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn(2+) at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37 degrees impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn(2+). 5. Both ammonium sulphate and heparin ;restart' the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn(2+) than of Mg(2+). 6. alpha-Amanitin abolishes the effect of ammonium sulphate and of heparin.     

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.     

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).