Cleavage of single strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1.
RNase H1 from Escherichia coli cleaves single strand RNA extending 3' from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9-17 nucleotides were designed to create overhanging single strand RNA regions extending 5' and 3' from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3' overhang region and not the 5' overhang region. RNase H digestion of the 3' overhang region resulted in digestion products with 5'-phosphate and 3'-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3' overhang region was not observed for a substrate that contained a 2'-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5' terminus of the 2'-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.
Lima
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Isis Pharmaceuticals, Inc., Carlsbad, California 92008, USA. wlima@isisph.com
Crooke
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D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D009692:Nucleic Acid Heteroduplexes; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*
D009692:Nucleic Acid Heteroduplexes; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI