DNA polymerase mechanism and fidelity: Using beta,gamma-bridging oxygen substituted dNTPs to study the mechanism of DNA polymerase beta.

摘要

A beta,gamma-bridging oxygen substituted dNTP toolkit comprising a variety of steric and electrostatic properties has been synthesized and used to investigate DNA polymerase beta. Surprisingly, when the logarithm of the presteady state kinetic rate of incorporation is plotted opposite the leaving group pKa4, rather than the predicted single line LFER, two lines are revealed, one containing only those substitutions with two halogens, and one for the remaining analogues. When the LFERs for dGTP and dTTP analogues, containing the same substitutions, are compared, there are large differences between the two, which is not expected a priori. In ternary crystal structures, only a single diastereomer is found for those compounds containing a CFX substitution, whereas the diastereomers of the remaining compounds are found in equal amounts. However, by a novel NMR detection method, both diastereomers were found to be used by the polymerase, though at different rates. Plotting the individual diastereomers on the LFERs shows that the R diastereomer is close to that of the mixture, while the S diastereomer is not on the line at all. These observations are easily explained by a three-dimensional, rather than the traditional two-dimensional, reaction coordinate diagram in which conformation and chemistry are coupled, rather than independent.