The effect of different divalent cations on the kinetics and fidelity of Bacillus stearothermophilus DNA polymerase.

Although Mg2+ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo, Mn2+ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg2+ or Mn2+ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg2+ or Mn2+ with BST DNA polymerase (BST pol), an A family DNA pol. We selected the metal ions based on whether they had previously been shown to be effective with other DNA pols. We found that Co2+ and Cd2+ were the only cations tested that could replace Mg2+ or Mn2+. When Co2+ was substituted for Mg2+, the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn2+, base selectivity was impaired compared to Co2+ and Cd2+. In addition, Co2+ and Mn2+ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co2+ and Mn2+ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B, for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3'-hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group, making it a highly effective nucleophile that can attack the α phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly, two metal ions are required for proofreading; one helps to lower the pKa of the attacking water molecule, and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all, of the effects of these different metal ions can be rationalized based on their intrinsic properties, which are tabulated in this review.

Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase.

Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA.

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

Polymerase and exonuclease activities in herpes simplex virus type 1 DNA polymerase are not highly coordinated.

The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.

Chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae.

Saccharopine reductase (SR) [saccharopine dehydrogenase (l-glutamate forming), EC 1.5.1.10] catalyzes the condensation of l-alpha-aminoadipate-delta-semialdehyde (AASA) with l-glutamate to give an imine, which is reduced by NADPH to give saccharopine. An acid-base chemical mechanism has been proposed for SR on the basis of pH-rate profiles and solvent deuterium kinetic isotope effects. A finite solvent isotope effect is observed indicating that proton(s) are in flight in the rate-limiting step(s) and likely the same step is limiting under both limiting and saturating substrate concentrations. A concave upward proton inventory suggests that more than one proton is transferred in a single transition state, likely a conformation change required to open the site and release products. Two groups are involved in the acid-base chemistry of the reaction. One of these groups catalyzes the steps involved in forming the imine between the alpha-amine of glutamate and the aldehyde of AASA. The group, which has a pK(a) of about 8, is observed in the pH-rate profiles for V(1) and V(1)/K(Glu) and must be protonated for optimal activity. It is also observed in the V(2) and V(2)/K(Sacc) pH-rate profiles and is required unprotonated. The second group, which has a pK(a) of 5.6, accepts a proton from the alpha-amine of glutamate so that it can act as a nucleophile in forming a carbinolamine upon attack of the carbonyl of AASA.

Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae.

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.