2 THERMOSTABLE TYPE-II RESTRICTION ENDONUCLEASES FROM ICELANDIC STRAINS OF THE GENUS THERMUS - TSP4C-I (ACN GT), A NOVEL TYPE-II RESTRICTION-ENDONUCLEASE, AND TSP8E-I, AN ISOSCHIZOMER OF THE MESOPHILIC ENZYME BGL-I (GCCNNNN/NGGC)/

Sixteen isolates of thermophilic bacteria from the genus Thermus, isol ated from neutral and alkaline hot water springs in the southwest regi on of Iceland, were tested for the presence of restriction endonucleas es. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two o f the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzyme s from these two Thermus isolates were partially purified and characte rized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the i nterrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme a ctivity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-strande d DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overh ang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic T ype II restriction endonuclease Bg/I(GCCNNNN/NGGC) [Lee, Clanton and C hirikjiam(1979) Fed. Proc. 28, 294], generating fragments with a three base 3'-OH overhang. However, unlike Bgl I, Tsp8E I exhibits consider able thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of t he extremely thermostable restriction endonucleases.