Elucidating the kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase B1

Biochemistry. 2009 Aug 11;48(31):7502-11. doi: 10.1021/bi9005336.


Transient-state kinetic techniques were used to resolve the kinetic mechanism of DNA polymerization catalyzed by an exonuclease-deficient mutant of Sulfolobus solfataricus P2 DNA polymerase B1 (PolB1 exo-). Here, we report the kinetic parameters of several elementary steps for the forward polymerization reaction. PolB1 exo- binds tightly to DNA (K(d)(DNA) = 1.8 nM) and a correct incoming nucleotide (apparent K(d)(dTTP) = 11 microM). Moreover, several lines of kinetic evidence suggested that correct nucleotide incorporation catalyzed by PolB1 exo- was limited by a protein conformational change which precedes the chemistry step. The utilization of an "induced fit" mechanism by PolB1 exo- was supported by the following: a small, alpha-thio elemental effect of 1.5, varying DNA dissociation rates for the binary complex (0.043 s(-1)) as well as ternary complexes before (0.18 s(-1)) and after (0.0071 s(-1)) a conformational change, a greater amplitude for the pulse-chase than the pulse-quench reaction, and an activation energy barrier of 38 kcal/mol which is greater than the predicted values of phosphodiester bond formation both in solution and within a polymerase active site. Lastly, PolB1 exo- exhibited a low processivity value of 15, thereby suggesting a protein cofactor confers this replicative DNA polymerase with higher processivity in vivo.

Publication types

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

MeSH terms

  • Catalysis
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / metabolism*
  • DNA, Archaeal / chemistry
  • DNA, Archaeal / metabolism
  • Exodeoxyribonucleases / deficiency
  • Exodeoxyribonucleases / genetics
  • Kinetics
  • Polymers / chemistry
  • Polymers / metabolism
  • Sulfolobus solfataricus / enzymology*


  • DNA, Archaeal
  • Polymers
  • DNA Polymerase beta
  • Exodeoxyribonucleases
  • exodeoxyribonuclease I