Multiple conformational changes in the mechanism of dna polymerase beta

X. Zhong, Smita Patel, B. G. Werneburg, M. D. Tsai

Research output: Contribution to journalArticle

Abstract

Two phases were observed in the stopped-flow fluorescence assay for the correct nucleotide dTTP incorporation using a DNA substrata containing 2aminopurine. The results from Mg2+ and dTTP concentration dependence of the observed rate constants and from the experiment with a DNA substrata containing a dideoxy-nucleotide terminated primer indicated that both phases were attributed to conformational changes. 0nly the fast phase was observed with the exchange-inert beta, gamma-CrdTTP complex in the absence of Mg2+, but both phases were observed in the presence of Mg2+, These evidence indicated that the MgdTTP binding induces the first conformational change and the binding of the catalytic Mg2+ at the second site further induces the second conformational change. The observed rates for incorrect nucleotide incorporation were consistent with the assumption that the first conformational change is associated with base-pairing. The bad base-pair geometry which resulted from incorrect MgdNTP binding may cause the alpha phosphate of MgdNTP to deviate from the right position for the catalytic Mg2+ binding and greatly raise the kinetic energy barrier for the second rate-limiting conformationai change, which could provide a major approach to achieve fidelity.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997
Externally publishedYes

Fingerprint

Nucleotides
nucleotides
Base Pairing
DNA
Energy barriers
Kinetic energy
Rate constants
Assays
Fluorescence
Phosphates
fluorescence
phosphates
kinetics
Geometry
energy
assays
Experiments
thymidine 5'-triphosphate
geometry

All Science Journal Classification (ASJC) codes

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Zhong, X. ; Patel, Smita ; Werneburg, B. G. ; Tsai, M. D. / Multiple conformational changes in the mechanism of dna polymerase beta. In: FASEB Journal. 1997 ; Vol. 11, No. 9.
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Multiple conformational changes in the mechanism of dna polymerase beta. / Zhong, X.; Patel, Smita; Werneburg, B. G.; Tsai, M. D.

In: FASEB Journal, Vol. 11, No. 9, 01.12.1997.

Research output: Contribution to journalArticle

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AU - Zhong, X.

AU - Patel, Smita

AU - Werneburg, B. G.

AU - Tsai, M. D.

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AB - Two phases were observed in the stopped-flow fluorescence assay for the correct nucleotide dTTP incorporation using a DNA substrata containing 2aminopurine. The results from Mg2+ and dTTP concentration dependence of the observed rate constants and from the experiment with a DNA substrata containing a dideoxy-nucleotide terminated primer indicated that both phases were attributed to conformational changes. 0nly the fast phase was observed with the exchange-inert beta, gamma-CrdTTP complex in the absence of Mg2+, but both phases were observed in the presence of Mg2+, These evidence indicated that the MgdTTP binding induces the first conformational change and the binding of the catalytic Mg2+ at the second site further induces the second conformational change. The observed rates for incorrect nucleotide incorporation were consistent with the assumption that the first conformational change is associated with base-pairing. The bad base-pair geometry which resulted from incorrect MgdNTP binding may cause the alpha phosphate of MgdNTP to deviate from the right position for the catalytic Mg2+ binding and greatly raise the kinetic energy barrier for the second rate-limiting conformationai change, which could provide a major approach to achieve fidelity.

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