Mismatched and matched dNTP incorporation by DNA polymerase beta proceed via analogous kinetic pathways.

Abstract:

While matched nucleotide incorporation by DNA polymerase beta (Pol beta) has been well-studied, a true understanding of polymerase fidelity requires comparison of both matched and mismatched dNTP incorporation pathways. Here we examine the mechanism of misincorporation for wild-type (WT) Pol beta and an error-prone I260Q variant using stopped-flow fluorescence assays and steady-state fluorescence spectroscopy. In stopped-flow, a biphasic fluorescence trace is observed for both enzymes during mismatched dNTP incorporation. The fluorescence transitions are in the same direction as that observed for matched dNTP, albeit with lower amplitude. Assignments of the fast and slow fluorescence phases are designated to the same mechanistic steps previously determined for matched dNTP incorporation. For both WT and I260Q mismatched dNTP incorporation, the rate of the fast phase, reflecting subdomain closing, is comparable to that induced by correct dNTP. Pre-steady-state kinetic evaluation reveals that both enzymes display similar correct dNTP insertion profiles, and the lower fidelity intrinsic to the I260Q mutant results from enhanced efficiency of mismatched incorporation. Notably, in comparison to WT, I260Q demonstrates enhanced intensity of fluorescence emission upon mismatched ternary complex formation. Both kinetic and steady-state fluorescence data suggest that relaxed discrimination against incorrect dNTP by I260Q is a consequence of a loss in ability to destabilize the mismatched ternary complex. Overall, our results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol beta originates from destabilization of the mismatched closed ternary complex and chemical transition state.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Nucleotide Analogs / Template Lesions, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Pol beta I260Q Roettger MP2008 Kd 7.1uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State (T template)
Pol beta I260Q Roettger MP2008 Kd 49uM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State (T template)
Pol beta I260Q Roettger MP2008 Kd 2.2uM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State (C template )
Pol beta I260Q Roettger MP2008 Kd 45uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State (C template)
Human Pol beta Roettger MP2008 Kd 6.8uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State (T template)
Human Pol beta Roettger MP2008 Kd 489uM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State (T template)
Human Pol beta Roettger MP2008 Kd 2.2uM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Steady State (C template)
Human Pol beta Roettger MP2008 Kd 227uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Steady State (C template)

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