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 pK a of the hydroxyl group, making it a highly effective nucleophile that can attack the ␣ 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 pK a 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.All DNA polymerases (DNA pols) 2 require Mg 2ϩ or Mn 2ϩ for primer extension and for excision of incorrectly incorporated dNTPs via intrinsic 3Ј35Ј exonuclease activity (1-5). A two-metal-ion mechanism is used by all DNA pols to catalyze nucleotide addition to a growing primer strand (6). Although DNA polymerases employ the physiologically relevant Mg 2ϩ , other divalent metal ions can substitute for Mg 2ϩ , although they tend to reduce the fidelity of DNA replication (7-10). The effect of metal ion cofactors on the fidelity of DNA replication has been studied for various DNA pols including E. coli DNA pol I (11), AMV DNA pol (12), Klenow fragment of E. coli DNA pol I (13), T4 pol (7), T7 pol (7), human pol ␣ (7), pol ␤ (7), and Dpo4 (8). Some metal ions have been shown to be mutagens and carcinogens probably because they reduce the base selectivity of DNA pols (7,8,(11)(12)(13)(14)(15). Different divalent cations influence fidelity check points in the minimal kinetic scheme for the nucleotidyl transfer reaction (Scheme 1). Cations that can substitute for Mg 2ϩ affect DNA pols by: 1) altering the groundstate binding affinity of incoming dNTPs to pol⅐DNA binary complexes (16); 2) decreasing base selectivity by promoting misincorporation during primer extension (8); 3) decreasing the rate of base excision (17); 4) altering primer extension past a mismatch at the primer-template (P/T) terminus (17). This review will address the way various metal ions increase misincorporation based on their physical properties. Our emphasis will be on th...

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.

Although Mg2+ is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn2+ can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg2+ or Mn2+ can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg2+ or Mn2+ with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co2+, and to a lesser extent Ni2+, were the only cations among those tested besides Mg2+ and Mn2+ that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co2+, relative to that of Mg2+. The incorporation efficiencies of incorrect dNMPs increased by 2–17-fold with Co2+, relative to that with Mg2+ depending on the incoming dNTP. Base selectivity was reduced even further with Mn2+ compared to that observed with Co2+. Substitution of Mn2+, Co2+, or Ni2+ for Mg2+ reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co2+ and Mn2+ were better able to extend a primer past a mismatch than Mg2+. Finally, Co2+ and Mn2+ enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer–template complexes.

During DNA synthesis base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base-pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the Penultimate Base-pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The incorporation efficiency for incoming dNTPs opposite an abasic site varied between 2 and 210 fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB’s interface and that the kinetic parameters for dNMP incorporations were consistent with specific features of base-stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated so that these surrounding water molecules become ordered and remain on the PB’s surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex and the incorporation efficiency of incoming dNTPs is reduced.

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.