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 language||English (US)|
|Publication status||Published - Dec 1 1997|
All Science Journal Classification (ASJC) codes
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology