Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase 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. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. 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 at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.
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INSERM U386, IFR Pathologies Infectieuses, Université Victor Segalen-Bordeaux II, 143 rue Léo Saignat, 33076 Bordeaux cédex, France.
Toulme
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D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D010053:Ovary / Q000201:enzymology; D012313:RNA / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D014982:Xenopus laevis
D012313:RNA; D004247:DNA; C091139:ribonuclease HII; D016914:Ribonuclease H; C078856:ribonuclease HI