Study on the characteristic mode of action of T4 DNA ligase

Abstract

The enzyme T4 DNA ligase which catalyzes the formation of a phosphodiester linkage between DNA chains was isolated from E.coli λT4 lig lysogen, and its mode of action for cohesive and blunt end termini was characterized by Eco RI, Pst I, Hind III and Sma I-generated pUC 9 as substrates. The isolated T4 DNA ligase has molecular weight of 68,000±3,000 as judged by 12% polyacrylamide gel electrophoresis containing 0.1% sodium dodecyl sulfate and the degree of joining activity was optimum at pH7.4-7.8 in the presence of 10 mM $Mg^{++}$, 10mM DTT. The optimum concentration of ATP was 1.0 mM for cohesive end ligation, 0.2 mM for blunt end ligation. Both reaction were inhibited by monovalent cations such as $Na^+$, $K^+$, $ NH^+_4$ and $Cs^+$, by polyamines, spermine and spermidine, and by phosphate. Blunt end ligation was more sensitive to these effectors. Optimum temperature and ligation efficiency of the enzymic reaction was dependent to the base composition of DNA fragments generated by restriction endonucleases. Blunt end joining showed a non-linear dependency on enzyme concentration and required about 40-fold of enzyme in cohesive end ligation. On the while, the predominant products of the enzymic ligation were gained in either linear concatemers or covalently closed circular forms appropriate DNA concentration, reaction time and enzyme amounts.