A computational study of the hydrolysis of dGTP analogues with halomethylene-modified leaving groups in solution: implications for the mechanism of DNA polymerases.


DNA polymerases make up a family of enzymes responsible for regulating DNA replication and repair, which in turn maintains the integrity of the genome. However, despite intensive kinetic, crystallographic, and computational studies, elucidation of the detailed enzymatic mechanism still presents a significant challenge. We recently developed an alternative strategy for exploring the fidelity and mechanism of DNA polymerases, by probing leaving group effects on nucleotidyl transfer using a series of dGTP bisphosphonate analogues in which the beta,gamma-bridging oxygen was replaced by a series of substituted methylene groups (X = CYZ, where Y and Z = H, halogen, or another substituent). Pre-steady state kinetic measurements of DNA polymerase-catalyzed incorporation of correctly base paired (R) and mispaired (W) analogues demonstrated a strong linear free energy relationship (LFER) between the polymerase rate constant (k(pol)) and the highest pK(a) of the free bisphosphonic acid corresponding to the leaving group. However, unexpectedly, the data segregated into two distinctly different linear correlations depending on the nature of the substituent. The discrepancy between the two lines was considerably greater when the dGTP analogue formed an incorrect (G.T) rather than a correct (G.C) base pair, although the reason for this phenomenon remains unexplained. Here, we have evaluated the complete free energy surfaces for bisphosphonate hydrolysis in aqueous solution and evaluated the corresponding LFER. Our study, which employs several alternative solvation models, finds a split of the calculated LFER for the mono- and dihalogen compounds into two parallel lines, reflecting their behavior in the polymerase-catalyzed condensation reaction. We suggest that the division into two linear subsets may be a generalized solvation phenomenon involving the overall electrostatic interaction between the substrates and their surroundings and would also be observed in polar solvents in the absence of the enzyme, if the reaction in solvent is in fact identical to that of the enzyme. However, the amplified differences between the LFER lines for the incorporation of matched and mismatched deoxynucleotides probably reflects the differences in the electrostatic interaction between the TS charges in the polymerase active site. An understanding of the mechanism of this reaction in solution could thereby provide a steppingstone for understanding the factors governing the fidelity of DNA polymerases.




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