DNA polymerase beta: multiple conformational changes in the mechanism of catalysis.

Abstract:

Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase beta (Pol beta), 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 dTTPalphaS 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.

Polymerases:

Topics:

Status:

new topics/pols set partial results complete validated

Results:

No results available for this paper.

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.