Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is anRNase HII

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Sub strates were either blunt-ended, or presented RNA or RNA overhangs. The hyd rolysis profiles obtained at early times of the digestion showed a good cor relation between the class of RNase H, either type I or II assigned accordi ng to biochemical parameters, whatever the organism. Consequently, the patt ern of primary cuts can be considered as a signature of the predominant RNa se H activity. For a given sequence, hydrolysis profiles obtained are simil ar, if not identical, for either blunt-ended:substrates or those presenting overhangs. However, profiles showed variations depending on the sequence u sed. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two Simultaneous primary cuts a t 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles furt her confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.