A new paradigm of DNA synthesis: three-metal-ion catalysis

Abstract

Enzyme catalysis has been studied for over a century. How it actually occurs has not been visualized until recently. By combining in crystallo reaction and X-ray diffraction analysis of reaction intermediates, we have obtained unprecedented atomic details of the DNA synthesis process. Contrary to the established theory that enzyme-substrate complexes and transition states have identical atomic composition and catalysis occurs by the two-metal-ion mechanism, we have discovered that an additional divalent cation has to be captured en route to product formation. Unlike the canonical two metal ions, which are coordinated by DNA polymerases, this third metal ion is free of enzyme coordination. Its location between the α- and β-phosphates of dNTP suggests that the third metal ion may drive the phosphoryltransfer from the leaving group opposite to the 3'-OH nucleophile. Experimental data indicate that binding of the third metal ion may be the rate-limiting step in DNA synthesis and the free energy associated with the metal-ion binding can overcome the activation barrier to the DNA synthesis reaction.

Fig. 1

DNA synthesis reaction. a Chemistry of DNA synthesis reaction. b The structure of Pol η-DNA-dNTP (enzyme-substrate) complex (ES) replete with the canonical two metal ions. The B-site metal ion comes along with dNTP. Binding of the A site metal ion is greatly enhanced by a correct incoming dNTP, which forms a Watson–Crick base pair with the template base. With two canonical metal ions bound, the reactants 3′-OH and the α-phosphate of dNTP are perfectly aligned, but without the C-site metal ion no reaction can take place. c DNA Pol η in the enzyme-product complex (EP). The third metal ion occupies the C site and is coordinated by oxygen atoms from product DNA and pyrophosphate. The other four coordination ligands are water molecules, one of which may donate a proton to the pyrophosphate leaving group. d Co-existing ES and EP complexes of DNA Pol η. The third metal ion would clash with dNTP and is incompatible with the ES complex. The largest changes between ES and EP are the scissile phosphorus and the sugar pucker of the 3′-end nucleotide. e Superposition of DNA Pol η (purple Mg²⁺) and Pol β (pink and green Mn²⁺) in the enzyme-product complexes (EP). Despite different tertiary structures, the third metal ion (Mn²⁺) is also found in DNA Pol β in the same coordination geometry as in Pol η

Fig. 2

Comparison of the initiation of phosphoryltransfer in two- versus three-metal-ion catalysis. a In three-metal-ion catalysis, the C-site metal ion initiates the reaction by breaking the existing phosphodiester bond in dNTP and thus drives the phosphoryltransfer reaction. A well-aligned native 3′-OH is required for capture of the C-site metal ion and its deprotonation is a result of the reaction. b In two-metal-ion catalysis, the reaction starts by de-protonation of the 3′-OH (nucleophile), which activates nucleophilic attack and leads to breakage of the existing phosphodiester bond in dNTP

Fig. 3

Modification of the transition-state theory. a The transition state theory suggests (1) a quasi-equilibrium between the substrate and transition state, which have the same chemical components, and (2) enzyme accelerating reaction rate by stabilizing transition state and thus reducing the energy barrier. The uncatalyzed and enzyme-catalyzed reaction processes are shown in grey and blue, respectively. We find that the canonical two Mg²⁺ ions are insufficient to support DNA synthesis. b Finding a third Mg²⁺ ion necessary for catalysis leads us to propose a new paradigm. The enzyme helps reaction substrates to capture the additional divalent cation and obtain necessary energy for transition from the substrate to product state