Ribonuclease H of calf thymus: substrate specificity, activation, inhibition.
When the action of highly purified specimens of ribonuclease H (hybrid nuclease; RNA-DNA hybrid ribonucleotidohydrolase; EC 3.1.4.34) of calf thymus on a wide selection of homopolymer hybrids was studied, the extent, and even the occurrence, of hydrolysis was found to be governed by the interplay of several factors: the composition of the ribo strand, the length of the deoxyribo strand, and the nature of the activating metal. Mn2+ activates the enzymic cleavage of all hybrid combinations, Mg2+ only of those containing purine ribo strands, Co2+ only of poly(A) hybrids. A 1:1 hybrid of phage f1 DNA and RNA is, however, split in the presence of any of these activators. Hybrids with deoxyribo tetranucleotides can still be cleaved, but not with dinucleotides. The behavior of hybrids containing covalently linked runs of ribo and deoxyribopolynucleotides was studied with the hybrid poly(dT)-poly(A)7-(dA)X]. This hybrid is attacked by ribonuclease H so that the bulk of the resulting poly(dA) still retains one covalently linked riboadenylic acid end group, whereas a small proportion carries a ribo dinucleotide. Inhibition studies showed that ribonuclease H is inactivated irreversibly by pretreatment with S-adenosylmethionine at 35 degrees, but not at 0 degrees. S-Adenosylhomocysteine also is inhibitory, but not irreversibly; also it is essentially limited to the inhibition of the cleavage of purine ribo strands. When the enzyme is exposed simultaneously to both inhibitors, irreversible inactivation is diminished considerably.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004279:DNA, Viral / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013950:Thymus Gland / Q000201:enzymology
D002413:Cations, Divalent; D004279:DNA, Viral; D011089:Polydeoxyribonucleotides; D012367:RNA, Viral; D011061:Poly A; D011071:Poly T; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D012260:Ribonucleases