Proofreading hydrolysis reactions

The 3 -5 proofreading exonuclease is called into action when an incorrect dNMP is added to the 3 terminus. The 3 -5 exonuclease is located in a separate domain with a distinct active site. The chemical reaction of the exonuclease proceeds by hydrolysis (see Fig. lb), but it is remarkably similar to the polymerase reaction. Specifically, two metal ions catalyze the reaction metal ion A activates water to form a hydroxyanion nucleophile that attacks the phosphodiester bond of the 3 terminal mismatched nucleotide. Metal B stabilizes the developing charge on the dNMP leaving group. 

Figure 1 Chemical mechanism of DNA polymerase and 3 -5 exonuclease, (a) DNA polymerase reaction. The enzyme chelates two metal Ions using three aspartic acid residues (only two are shown). Metal ion A abstracts the 3 hydroxyl proton of the primer terminus to generate a nucleophile that attacks the a-phosphate of an incoming dNTP substrate. The phosphoryl transfer results In production of a pyrophosphate leaving group, which is stabilized by metal Ion B. (b) The 3 -5 exonuclease proofreading activity is located in a site that is distinct from the polymerase site yet it uses two-metal-ion chemistry similar to DNA synthesis. The reaction type is hydrolysis in which metal ion A activates water to form the hydroxy anion nucleophile. Nucleophile attack on the phosphate of the mismatched nucleotide releases it as dNMP (dGMP in the case shown).

The hydrolysis of GTP is coupled to the operation of many of the factors that control initiation, elongation and termination of protein synthesis in order to provide directionality and specificity to these processes [45]. The free energy of hydrolysis of ATP is utilized indirectly to provide specificity in proofreading processes, such as the cleavage of incorrectly activated amino acids on aminoacyl-tRNA or aminoacyl-AMP molecules [46]. GTP hydrolysis in the reaction cycle initiated by the... 

Reversible bond formation is utilized by living organisms during the first step of replication as a primary means of proofreading via the thermodynamic selection of the lowest energy base pair (i.e., correct Watson-Crick complement) (Scheme 1). The reversible condensation of the nucleoside triphosphate is followed by hydrolysis of the pyrophosphate product to kinetically trap the newly formed phosphodiester bond (Scheme 1). In emulating this two-step process, we maintain that both the initial step that exploits the stability of template association, and the subsequent reaction that traps the thermodynamically-favored product are necessary for high-fidelity repUcation. 

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