| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
|
|---|
| Research Abstract |
RNA and DNA polymerases are structurally similar and utilize similar enzymatic mechanisms of catalysis. However, these enzymes efficiently discriminate between ribonucleic acid, deoxyribonucleic acid, ribonucleotides and deoxyribonucleotides, such that they achieve a high degree of specificity for substrate and template. To elucidate the discrimination mechanisms, we have examined an O helix mutants of Taq pol I, A661E, for its ability to discriminated between ribonucleotides and deoxyribonucleotides. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less well than wild-type ; discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP and rGTP, respectively. The data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleotide, dGTP, but they are impared in their ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and depxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O helix and the terminal two phosphate groups in the primer strand. Our data have also shown that efficiency in phospheryl transfer reaction and replication fidelity, which share the same catalytic site, are capable to get separated in a DNA polymerase.
|
