TSP491 (ACGT-DOWN-ARROW), A THERMOSTABLE NEOSCHIZOMER OF THE TYPE II RESTRICTION ENDONUCLEASE MAEII (A-DOWN-ARROW-CGT), DISCOVERED IN ISOLATES OF THE GENUS THERMUS FROM THE AZORES, ICELAND AND NEW ZEALAND

摘要

One hundred and forty eight isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity, An isolate (SM49) from the island of Sao Miguel, in the Azores, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Tsp49I) has been partially purified from this isolate and the recognition and cleavage site determined, Tsp49I recognizes the four base sequence ACGT, which is the same as the recognition sequence of the mesophilic Type II restriction endonuclease MaeII, However, unlike MaeII, which cleaves DNA between the first and second base of the recognition sequence (A down arrow CGT), Tsp49I hydrolyses the phosphodiester bond in both strands of the substrate after the last base of the recognition sequence 5'-ACGT down arrow-3', producing four base 3'-OH overhangs (sticky ends), The enzyme has a pH optimum of 9.0, requires 2 mM MgCl2 for maximum activity and retains full enzyme activity following incubation for 10 min at temperatures up to 80 degrees C. Two further examples of the same restriction endonuclease specificity as Tsp49I were detected in Thermus isolates from Iceland (TsplDSI) and New Zealand (TspWAM8AI), The three MaeII neoschizomers, Tsp49I, TsplDSI and TspWAM8AI, exhibit similar pH optima, heat stabilities and MgCl2 requirements, but differ in their requirements for NaCl and KCl.