DNA ENZYMOLOGY ABOVE 100-DEGREES-C - TOPOISOMERASE V UNLINKS CIRCULARDNA AT 80-120-DEGREES-C

The widespread application of polymerase chain reaction and related te chniques in biology and medicine has led to a heightened interest in t hermophilic enzymes of DNA metabolism. Some of these enzymes are stabl e for hours at 100 degrees C, but no enzymatic activity on duplex DNA at temperatures above 100 degrees C has so far been demonstrated. Rece ntly, we isolated topoisomerase V from the hyperthermophile Methanopyr us kandleri, which grows up to 110 degrees C. This novel enzyme is sim ilar to eukaryotic topoisomerase I and acts on duplex DNA regions. We now show that topoisomerase V catalyzes the unlinking of double-strand ed circular DNA at temperatures up to 122 degrees C. In this in vitro system, maximal DNA unlinking occurs at 108 degrees C and corresponds to complementary strands being linked at most once. These results furt her imply that in the presence of sufficient positive supercoiling DNA can exist as a double helix even at 122 degrees C.