We report here the first use of a leaving-group substituent effect to gain a better understanding of the mechanism of DNA polymerase catalysis and fidelity. We have performed pre-steady-state single-turnover kinetic assays of DNA polymerase beta-directed incorporation of a series of dGTP analogs into single-nucleotide gapped DNA (template base C or T). These analogs are substituted at the beta-gamma bridging phosphate oxygen to create an increasingly electron withdrawing series {methylene-, monofluoromethylene-, oxo-, difluoromethylene-} of substituted-bisphosphonate leaving groups. Molecular dynamics simulations of steps in the pathway, both in a nonenzymatic solution-phase environment and in the protein, and x-ray diffraction on single crystals of the analogs in dideoxy-primer-terminated ternary complexes, were also performed. Experimentally, we find that the log(k_pol) versus leaving-group pK_a behavior shows a transition from near-zero linear slope (Brønsted beta) at low pK_a to modest negative slope at higher pK_a, for incorporation opposite both templates. This result, and the structural/computational data, can be explained in terms of a change in mechanism at sufficient stabilization of the bond-breaking step. TG-misincorporation of the methylene-substituted analog appears to be selected against to an approximately five-fold higher degree than the native substrate, due mainly to relatively poor analog binding opposite template base T; the other analogs exhibit TG-misincorporation frequencies close to that of native.