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

The widespread application of polymerase chain reaction and related techniques in biology and medicine has led to a heightened interest in thermophilic enzymes of DNA metabolism. Some of these enzymes are stable for hours at 100 degrees C, but no enzymatic activity on duplex DNA at temperatures above 100 degrees C has so far been demonstrated. Recently, we isolated topoisomerase V from the hyperthermophile Methanopyrus kandleri, which grows up to 110 degrees C. This novel enzyme is similar to eukaryotic topoisomerase I and acts on duplex DNA regions. We now show that topoisomerase V catalyzes the unlinking of double-stranded 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 further imply that in the presence of sufficient positive supercoiling DNA can exist as a double helix even at 122 degrees C.