Elucidating the kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase B1.
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.
Brown
Jessica A
JA
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Suo
Zucai
Z
D002384:Catalysis; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000172:deficiency / Q000235:genetics; D007700:Kinetics; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D019641:DNA, Archaeal; D011108:Polymers; D019951:DNA Polymerase beta; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I