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

Biochemistry
Michelle P RoettgerMing-Daw Tsai

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 intens...Continue Reading

References

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Citations

Mar 8, 2011·Journal of Theoretical Biology·Ping Xie
Jul 28, 2009·Biochimica Et Biophysica Acta·Jennifer Yamtich, Joann B Sweasy
Aug 13, 2014·DNA Repair·Rachelle J BienstockSamuel H Wilson
May 14, 2016·Physical Review. E·Pierre Gaspard
Nov 17, 2019·Nucleic Acids Research·Khadijeh S AlnajjarJoann B Sweasy
Jan 20, 2018·Physical Review. E·Pierre Gaspard

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Methods Mentioned

BETA
fluorescence spectroscopy
X-ray
Assay
Fluorescence
FRET

Software Mentioned

Sigma Plot
ImageQuant
Applied Photophysics
WEBMAXC STANDARD

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