Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase.
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.
Fiala
Kevin A
KA
Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Sherrer
Shanen M
SM
Brown
Jessica A
JA
Suo
Zucai
Z
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature
D019843:Archaeal Proteins; D009711:Nucleotides; D004247:DNA; D019951:DNA Polymerase beta