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

Nucleic Acids Res. 2008 Apr;36(6):1990-2001. doi: 10.1093/nar/gkn004. Epub 2008 Feb 14.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism
  • DNA / biosynthesis
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / metabolism
  • Kinetics
  • Nucleotides / metabolism
  • Protein Conformation
  • Sulfolobus solfataricus / enzymology*
  • Temperature*

Substances

  • Archaeal Proteins
  • Nucleotides
  • DNA
  • DNA Polymerase beta