Catalytic reactions of DNA polymerase I from E, coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 108 M-1 (kon = 105 M-1 S-1 and koff = 10-3 s-1), and decreased to 106 M-1 (k′on = 104 M-1S-1 and k′off = 10-2 S-1) for completely polymerized DNA, This is due to the 10-fold decrease in binding rate constant (kon) and 10-fold increase in dissociation rate constant (koff) for completed DNA strands, Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (kcat and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.
|ジャーナル||Chemistry - A European Journal|
|出版ステータス||出版済み - 8月 3 2001|
!!!All Science Journal Classification (ASJC) codes
- 化学 (全般)