DNA polymerase β: Multiple conformational changes in the mechanism of catalysis

Xuejun Zhong, Smita S. Patel, Brian G. Werneburg, Ming Daw Tsai

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Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase β (Pol β), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate- limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3' end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by the binding of the catalytic Mg2+ ion. The same biphasic kinetics with different rates were also observed with the thio analog dTTPαS and incorrect nucleotides dATP, dGTP, and dCTP. The structural nature for the two conformational changes has been discussed in relation to the available structural information of this enzyme. The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.

Original languageEnglish (US)
Pages (from-to)11891-11900
Number of pages10
Issue number39
StatePublished - Sep 30 1997
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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