Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases

Abstract

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...

Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

“…DNA polymerases such as the Taq and Bst DNA polymerases require a certain level of divalent metal-ions as cofactor for optimal performance27. Inhibitory effects on the enzymatic reaction by Mn 2+ and the selected dyes have to be checked first.…”

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors

“…Substitution mutations of the putative catalytic residues (45) as well as other charged residues in the RNase H domain (46) also affect HBV polymerase activity. In addition, HBV RNase H inhibitors might directly affect DNA strand elongation activity of HBV polymerase through chelating and/or altering the coordination geometry of the divalent metal cations in the HBV RT active site because HBV polymerase activity also requires divalent metal ions for catalysis (47).…”

Expression and Structural Analyses of Human DNA Polymerase θ (POLQ)

“…With physiological concentration of the catalytic Mg 2+ cation, pol θ does not have template-independent terminal transferase activity (Hogg et al, 2012), whereas high ratios of Mn 2+ over Mg 2+ appear to trigger template-independent extension (Kent, Chandramouly, McDevitt, Ozdemir, & Pomerantz, 2015; Kent, Mateos-Gomez, Sfeir, & Pomerantz, 2016). Mn 2+ has been shown to relax template specificity in many polymerases (Vashishtha, Wang, & Konigsberg, 2016). The ability of pol θ to extend single-stranded DNA could thus be largely explained by cycles of templating with other DNA molecules, slippage, and retemplating (Yousefzadeh et al, 2014; Wyatt et al, 2016).…”