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

• Published in: Nucleic acids research Vol. 28; no. 18; pp. 3674 - 3683
• Main Authors: Pileur, F, Toulme, J J, Cazenave, C
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 15.09.2000; Oxford University Press
• Subjects: Animals; Cattle; DNA - metabolism; Female; HeLa Cells; Humans; Hydrolysis; Mitochondria - enzymology; Mitochondria, Heart - enzymology; mitochondrial ribonuclease H; Nucleic Acid Hybridization; Ovary - enzymology; Ribonuclease H - metabolism; ribonuclease HI; ribonuclease HII; RNA - metabolism; Xenopus; Xenopus laevis
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/28.18.3674

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.