Nucleic Acids Research Advance Access published online on February 14, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn004
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Nucleic Acid Encymes |
Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase
1Department of Biochemistry, 2The Ohio State Biochemistry Program, 3The Molecular, Cellular & Developmental Biology Program and 4The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
*To whom correspondence should be addressed: Tel: +1 614 688 3706; Fax: +1 614 292 6773; Email: suo.3{at}osu.edu
Received August 6, 2007. Revised January 5, 2008. Accepted January 7, 2008.
Our previous publication shows that Sulfolobus solfataricus Dpo4 utilizes an ‘induced-fit’ mechanism to select correct incoming nucleotides at 37°C. Here, we provide a comprehensive report elucidating the kinetic mechanism of a DNA polymerase at a reaction temperature higher than 37°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°C increase in reaction temperature, suggesting that the fidelity of Dpo4 at 80°C is similar to that determined here at 56°C. Amazingly, the incorporation rate for correct nucleotides increased by 18 900-fold from 2°C to 56°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°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.
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