Based on the projected three dimensional equivalence of conserved amino acids in DNA polymerases. Arg 110 of MuLV RT is expected to be an important participant in the catalytic mechanism of MuLV RT. In order to obtain an evidence to support this proposition and to assess the functional importance of Arg 110. we carried out site directed mutagenesis of Arg 110 and replaced it with Lys, Ala and Glu. All three mutants exhibited a significant reduction in fc,al of polymerase activity that ranged from 3000 to 10.000 fold as compared to the wild type enzyme. The three mutant enzymes were extensively characterized with respect to kinetic parameters, ability to bind to template primer and catalyse nucleotide addition reaction and the mode of DNA synthesis. Results suggest that Arg 110 participates in the chemical step of phosphodiester bond formation in the catalytic mechanism of MuLV RT.
|Original language||English (US)|
|State||Published - Dec 1 1996|
All Science Journal Classification (ASJC) codes
- Molecular Biology