Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase.


Our previous publication shows that Sulfolobus solfataricus Dpo4 ...
Our previous publication shows that Sulfolobus solfataricus Dpo4 utilizes an 'induced-fit' mechanism to select correct incoming nucleotides at 37 degrees C. Here, we provide a comprehensive report elucidating the kinetic mechanism of a DNA polymerase at a reaction temperature higher than 37 degrees C in an attempt to determine the effect of temperature on enzyme fidelity and mechanism. The fidelity of Dpo4 did not change considerably with a 30 degrees C increase in reaction temperature, suggesting that the fidelity of Dpo4 at 80 degrees C is similar to that determined here at 56 degrees C. Amazingly, the incorporation rate for correct nucleotides increased by 18 900-fold from 2 degrees C to 56 degrees C, similar in magnitude to that observed for incorrect nucleotides, thus not perturbing fidelity. Three independent lines of kinetic evidence indicate that a protein conformational change limits correct nucleotide incorporations at 56 degrees C. Furthermore, the activation energy for the incorporation of a correct nucleotide was determined to be 32.9 kcal/mol, a value considerably larger than those values estimated for a rate-limiting chemistry step, providing a fourth line of evidence to further substantiate this conclusion. These results herein provide evidence that Dpo4 utilizes the 'induced-fit' mechanism to select a correct nucleotide at all temperatures.



Other Enzymatic Activities, Structure and Structure/Function, Fidelity


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