Proc. Nat. Acad. Sc. USA Vol. 73, No. 4, pp. April 1976 Biochemistry 1087-1091!, Ribonuclease H of calf thymus: Substrate specificity, activation, inhibition (hybrids of deoxyribo and ribo homopolymers/copolymer of deoxyribo. and riboadenylic acid/S-adenosylmethionine/ Sadenosylhomocysteine) JANNIS G. STAVRIANOPOULOS*, ANGELA GAMBINO-GIUFFRIDA, AND ERWIN CHARGAFF* Cell Chemistry Laboratory, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 Contributed by Erwin Chargaff, January 19,1976 ABSTRACT When the action of highly purified speci- years. Pancreatic deoxyribonuclease I, free of ribonuclease mens of ribonuclease H (hybrid nuclease; RNA-DNA hybrid (EC 3.1.4.5), was a Worthington product. ribonucleotidohydrolase; EC 3.1.4.34) of calf thymus on a wide selection of homopolymer hybrids was studied, the ex- SUBSTRATES tent, and even the occurrence, of hydrolysis was found to be governed by the interplay of several factors: the composition Homopolymers. The polydeoxynucleotides were synthe- of the ribo strand, the length of the deoxyribo strand, and the sized with the aid of the terminal deoxynucleotidyl transfer- nature of the activating metal. Mn2+ activates the enzymic ase of calf thymus (3), in the presence of Mg2+ ions for poly- cleavage of all hybrid combinations, Mg2+ only of those con- ions for and The taining purine ribo strands, Cot+ only of poly(A) hybrids. A (dA) and of Co2+ poly(dC) poly(dT). phage 1:1 hybrid of phage fi DNA and RNA is, however, split in the fI DNA-RNA hybrid (1:1) has been described before (4). presence of any of these activators. Hybrids with deoxyribo The 3H-labeled ribopolymers poly(U) and poly(C) were pre- tetranucleotides can still be cleaved, but not with dinucleo- pared and purified by a modification of a previous proce- tides. The behavior of hybrids containing covalently linked dure (5). The (dA)-(U), (dT)-(A), and (dG)-(C) hybrids were runs of ribo and deoxyribopolynucleotides was studied with made by mixing equimolar quantities of the complementary the hybrid poly(dT)'poly[(A dA)J. This hybrid is attacked homopolymers in 0.05 M Tris-HCI (pH 8.0); they were by ribonuclease H so that the bulk of the resulting poly(dA) still retains one covalently linked riboadenylic acid end stored at -20°. For the (dC)-(G) hybrids, the components group, whereas a small proportion carries a ribo dinucleo- were kept at 780 for 4 hr, then cooled overnight to 450, and tide. Inhibition studies showed that ribonuclease H is inacti- the hybrids were stored at 00. vated irreversibly by pretreatment with Sadenosylmethion- Radioactive poly(A) was made by the action of RNA poly- ine at 350, but not at 0°. S.Adenosylhomocysteine also is in- merase on poly(dT) in the presence of [3H]ATP (2600 hibitory, but not irreversibly; also it is essentially limited to or as described the inhibition of the cleavage of purine ribo strands. When cpm/nmol) [14C]ATP (7000 cpm/nmol) (1). the enzyme is exposed simultaneously to both inhibitors, irre- Radioactive poly(G) was made on a poly(dC) template. The versible inactivation is diminished considerably. mixture (7 ml) consisted of 0.05 M KCI, 3 mM MgCl2, 4 mM dithiothreitol, 2 mM [3H]GTP (3500 cpm/nmol), 1.2 mM We continue our study of ribonuclease H (hybrid nuclease; poly(dC), and 7 units/ml of RNA polymerase, in 0.05 M RNA-DNA hybrid ribonucleotidohydrolase; EC 3.1.4.34) of Tris-HCI of pH 8.5 (at 350). On incubation at 350 under calf thymus (1) by directing our attention to problems of argon, polymerization is very fast initially, but progresses substrate specificity, the influence of different metals on the slowly and nearly comes to a stop at 20 hr. The yield of course of substrate hydrolysis, and the specific inhibition of poly(G) varied widely, between 25 and 82% of the poly(dC) the enzyme. template employed. After addition of 14 ml of ethanol and storage overnight at 40, the precipitate was collected by cen- MATERIALS trifugation (10,000 X g, 10 min), and its solution in 2 ml of In addition to materials listed before (1) the following prepa- the Tris.HCI buffer was dialyzed for 3 days against 0.05 M rations were employed. Ribonucleoside diphosphates and S- KCI to remove traces of GTP. adenosylhomocysteine came from Sigma, S-adenosylmeth- Poly(G) was obtained by treating the poly(G)-poly(dC) ionine sulfate from Boehringer, oligodeoxynucleotides (di, hybrid with deoxyribonuclease in 0.02 M Tris-HCI (pH 7.5) tetra, octa) from Collaborative Research, Inc. Hydroxylapa- and 0.001 M MnCI2. After dialysis for 24 hr against the same tite was used as Bio-Gel HTP (Bio-Rad). Most enzymes em- buffer at room temperature, the Mn salt of poly(G) was col- ployed, including the highly purified thymus ribonuclease lected by centrifugation and taken up in 0.001 M EDTA. H, have been described before (1). Polynucleotide phospho- Oligoadenylate. Radioactive oligo(A) with a free terminal rylase (EC 2.7.7.8) was obtained as by-product during the 3' hydroxyl was prepared by partial hydrolysis of the hybrid isolation of RNA polymerase from Escherichia coil B (EC of poly(dT) with poly(['4C]A) (7000 cpm/nmol), described 2.7.7.6). The latter enzyme preparation was free of polynu- before, with the aid of ribonuclease H. The incubation mix- cleotide phosphorylase and of ribonuclease. The DNA poly- ture (8 ml) consisted of 25 mM MgCl2, 0.05 mM KCI, 250 merase (EC 2.7.7.7) of chicken embryo (2) had been stored 2 '4g/ml of bovine serum albumin, a quantity of the hybrid poly(dT)-poly(A) corresponding to 187.5 nmol/ml of ade- Abbreviations: AdoMet, S-adenosylmethionine; AdoHcy, S-adeno- nylic acid, 125 units/ml of ribonuclease H (1), with the en- sylhomocysteine. zyme activity determined in the presence of Mg2+, in 0.05 * Present address to which reprint requests should be sent: Cell M Tris-HCI of pH 8.0. The incubation mixture without the Chemistry Laboratory, The Roosevelt Hospital, 428 West 59th St., enzyme was brought to 350 and the reaction was then start- New York, N.Y. 10019. ed by the addition of the enzyme. After 15 min at 350, the 1087 Downloaded by guest on September 23, 2021 1088 Biochemistry: Stavrianopoulos et al. Proc. Nat. Acad. Sci. USA 73 (1976) mixture was adjusted to pH 2.0-2.5 with 1 M HCl, thus in- 0.5 M potassium phosphate (pH 6.8) of the hybrid poly(dT)- activating the enzyme, and left at this pH for 10 min. Fol- poly[(A)7-(dA),]. The balance of radioactivity (as nmol of lowing neutralization with 1 M Tris base and adjustment to [14C]adenylic acid) was: placed on a column, 489.4; eluted as 37.5 mM EDTA, the solution was applied to a column of 2 oligonucleotides, 88.3 (18.0%); eluted as polymer hybrid, ml of hydroxylapatite equilibrated with 0.15 M KCL. Elution 314.9 (64.3%). A portion, 17.6% of the radioactivity, was not was performed with potassium phosphate buffer of pH 6.8: eluted, presumably held back on the column as an enzyme- 0.025 M for the oligonucleotides and 0.3 M for the unhydro- polynucleotide complex. lyzed hybrid. The 0.025 M eluate was placed on a column of The 0.5 M potassium phosphate eluate of the polymer hy- 0.5 ml of DEAE-cellulose equilibrated with 0.05 M Tris.HCl brid was freed of phosphate by being passed through a Se- of pH 8.0.t The small oligonucleotides (n < 5) were eluted phadex G-25 column equilibrated with 0.1 M KCl or 0.2 M with 8 ml of 0.2 M KC1 (flow rate 4 ml/hr) and the oligonu- ammonium sulfate. cleotides of an average length of n = 7.2 were eluted with This hybrid was completely resistant to pancreatic ribonu- 0.4 M KCl at the same flow rate, the first 300 Mtl of eluate clease, as shown by the amount of radioactivity precipitable being discarded. Four fractions each of 0.5 ml were collect- with 10% trichloroacetic acid at zero time and after enzyme ed, of which the first three contained 4.561 X 106 cpm or treatment. In parallel experiments (in each: 55 gl solution, 43.4% of the poly([14C]A) submitted to enzymic hydrolysis; 0.1 M KCI, 0.01 M EDTA, pH 7.5, 15,000 cpm, 100 Mug of ri- the fourth eluate fraction corresponded to only 3% of the bonuclease, 350, 30 min), the radioactivity precipitable at original radioactivity and was discarded. The proportion of zero time was 14,930 and 15,050 cpm, and after ribonucle- radioactivity precipitable by trichloroacetic acid was deter- ase treatment, 15,070 and 14,540 cpm. mined separately in 0.25 Ml samples: in eluate fractions I and II it was only 1.8%; in fractions III, 10%; and in fraction IV, RESULTS 14.3%. The average length of the oligo(A) preparation was Factors Influencing the Action of Ribonuclease H on estimated by alkaline hydrolysis (0.66 M LiOH, 370, 18 hr) Homopolymer Hybrids. The base composition of the ribo and determination of adenosine, 2'(3')-adenylic acid, and strand, the length of the deoxyribo strand, the nature and adenosine 3',5'-diphosphate (paper chromatography in 1- the concentration of the metal activator, and also the salt propanol/concentrated ammonia/water, 11:2:7, vol/vol/ concentration of the medium (left out of consideration here), vol).