Ribonuclease H of Calf Thymus: Substrate Specificity, Activation, Inhibition (Hybrids of Deoxyribo and Ribo Homopolymers/Copolymer of Deoxyribo

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). Since the length was found as 7.2 (based on pAp) or 7.5 all these determine, though to a varying degree, the course (based on adenosine), this preparation is designated as (A)7.* and the outcome of the enzymic action. Hybrid of Poly[(AHdA~J and Poly(dT). This copolymer Base Composition of Ribo Strand. Poly(dT)-poly(A), in hybrid was made by the action of the DNA polymerase of the presence of Mg2+, is the best substrate, resembling the fi chicken embryo (2) obi the hybrid of poly(dT) with the 14C- DNA-RNA hybrid (1:1) discussed previously (1) in its hy- labeled ribo oligomer p(A)7 in the presence of dATP: The drolysis requirements. Poly(dC)-poly(G) is attacked much incubation mixture (12 ml) consisted of 1.25 mM MnCl2, 83 more slowly, requiring four times as long for the same de- mM KC1, 1.7 mM dithiothreitol, 400,uM dATP, 500,MM po- gree of splitting as poly(dT)-poly(A) in the presence of ly(dT), 50 MuM(A)7, 33 units/ml of DNA polymerase, and Mn2+. The results summarized in Table 1 illustrate the vari- 250 ug/ml of bovine serum albumin, in 0.05 M Tris.HCl of ety of conditions governing the enzymic hydrolysis of the pH 8.0. In order to facilitate a uniform distribution of the ol- homopolymer hybrids under study. The fl hybrid men- igoadenylate on the poly(dT) present in a 10-fold excess, the tioned before is included for comparison. The normalization following procedure was employed. A solution of the (Ah of all results, required for comparison, is also explained in and the poly(dT) in 7 ml of 0.116 M KCl was kept at 700 for this table. 10 min and then slowly brought to room temperature, the Length of Deoxyribo Strand. As is also shown in Table 1, rest of the additives (minus enzyme) in 4.9 ml of buffer was the degradation velocity rises with the increasing length of admixed in an argon atmosphere, and the mixture was the deoxyribo strand. It is, however, remarkable that even cooled to 00. Then the enzyme was added and incubation deoxyribonucleotides as short as tetra offer sufficient sup- was performed at 200 for 12 hr (compare Table 1 in ref. 4). port for the ribo strand to be attacked by the hybrid-specific The radioactivity and its precipitability with trichloroace- enzyme, since the temperature of the enzyme reaction, 350, tic acid at zero time and at the end of the incubation were presumably lies above the melting temperatures of the re- measured in duplicate in 50 gl portions. Per 50 Ml portion spective double structures (6). It would not be surprising if there were 17,181 cpm, of which 1226 (7.1%) were precipi- the stability of the hydrogen-bonded alignment of the two table at zero time and 14,275 (83.1%) were precipitable at strands were, in fact, increased by the enzyme as it proceeds the end of the reaction. to degrade the hybrid. The study of the effect of deoxyribo In order to remove unincorporated oligonucleotides, the tetranucleotides, of which all four representatives could be incubation mixture was adjusted to 2.0 mM EDTA and to examined (Exps. 1, 3, 6, and 9 in Table 1), offers an opportu- pH 7.0 by M Tris.HCI and then placed on a column of 1.5 nity of comparing the influence of the particular base on the ml of hydroxylapatite equilibrated with 0.15 M KCI. The hydrolysis of the complementary ribo strand. The efficiency unpolymerized oligonucleotides were eluted with 0.05 M decreases in the following order: (dA)4.poly(U); (dG)4. potassium phosphate of pH 6.8, followed by the elution with poly(C); (dC)4.poly(G); (dT)4.poly(A), when Mn2+ is the activator. Pyrimidine ribo strands are broken more readily t Microcolumns are prepared conveniently by using the polypro- than purine strands. The only deoxyribo dinucleotide avail- pylene tip of a 1 ml Eppendorf automatic pipette closed with a able, d(pApA), did not support the enzymic degradation of small plug of glass wool. the complementary polyribo strand (Exp. 5). The average chain length of these products is, of course, not en- Divalent Metal Ions. The differential effects are some- tirely the same in different experiments. For instance, in an assay in which poly([3H]A)-poly(dT) was treated with ribonuclease H what baffling. Mn2+ activates the attack on all hybrid com- by a somewhat different procedure, the resulting oligo(A) had an binations; Mg2+, only on those containing purine ribo average chain length of 8.9. strands; Co2+, only on poly(A) hybrids, but less well than Downloaded by guest on September 23, 2021 Biochemistry: Stavrianopoulos et al. Proc. Nat. Acad. Sci. USA 73 (1976) 1089

Table 1. Degradation efficiency of different hybrid Table 2. Effects of metal ions on ribonuclease H* substrates by ribonuclease H* Metal ion (mM) Components of equi- Effect of metal 100% molar hybrid on % degradation Components of Inhi- Deoxyribo Ribo Mg2+ C02+ Mn2+ equimolar hybrid Optimum activity bition 1 (dT)4 Poly(A) 25 2.1 6.3 Deoxyribo Ribo Mn2+ Mg2+ C0o2+ Co2+ 2 Poly(dT) Poly(A) 100 70 40 (dT)4 Poly(A) 1,5 10 2.5 3 (dC)4 Poly(G) 7.8 Inhib. 6.4 Poly(dT) Poly(A) 1.5 25 20 4 Poly(dC) Poly(G) 12.5 Inhib. 10.4 (dC)4 Poly(G) 0.4 12 8 5 (dA)2 Poly(U) Inact. Inact. Inact. Poly(dC) Poly(G) 0.4 10 8 6 (dA)4 Poly(U) Inact. Inhib. 17.5 (dA)4 Poly(U) 10 Inact. 1.5 7 (dA), Poly(U) Inact. Inhib. 28.2 (dA)s Poly(U) 10 Inact. 1.5 8 Poly(dA) Poly(U) Inact. Inhib. 62.5 Poly(dA) Poly(U) 1.5 Inact. 1.5 9 (dG)4 Poly(C) Inact. Inhib. 12.5 (dG)4 Poly(C) 7.5 Inact. 1 10 fl DNA RNA fl DNA RNA tran- tran- script 66.5 61.7 54.3 script 1.5 25 20 * Each assay sample (120 ul) consisted of 0.05 M Tris-HCl (pH *The experimental conditions were those indicated in Table 1, 8.0), of hybrid substrate corresponding to 83.3 MM with respect to with the enzyme and salt concentrations specified there. The the ribo moiety, of 0.4 mg/ml of bovine serum albumin, and of concentrations of metal activators or inhibitors varied, however, the additions specified below for each experiment. The indicated over the range shown here. enzyme concentrations were so chosen as to permit a 70-80% degradation of the ribo strand. The incubation was at 350 for 15 min. Exp. 1: 10 mM MgCl2, 40 ng of enzyme, 0.025 M KQl; 1.5 ml; 0.05 M Tris-HUl, pH 8.0; 0.025 M MgC12; 0.05 M KCl; mM MnCl2, 135 ng of enzyme, 0.1 M (NH4)2S04; 2.5 mM CoC12, 500 .g of bovine serum albumin; copolymer hybrid corre- 420 ng of enzyme, 0.2 M KCl.-Exp. 2: 25 mM MgCl2, 10 ng of sponding to 51.4 nmol of riboadenylic acid; and 500 units of enzyme, 0.05 M KQl; 1.5 mM MnCl2, 20 ng of enzyme, 0.2 M ribonuclease H.-Exp. 2: total volume 1.3 ml; 0.05 M Tris- (NH4)2SO4; 20 mM CoC12, 13 ng of enzyme, 0.35 M KCl.-Exp. 3: 15 mM MgCl2, 120 ng of enzyme; 0.25 mM MnCl2, 145 ng of HC1, pH 8.0; 2.0 mM MnCl2; 0.2 M (NH42S04; 500 ,ug of enzyme.-Exp. 4: 10 mM MgCl2, 70 ng of enzyme, 0.05 M KQl; bovine serum albumin; copolymer hybrid corresponding to 0.25 mM MnCl2, 80 ng of enzyme, 0.15 M KCl.-Exps. 5 and 6: 53.3 nmol of riboadenylic acid; and 500 units of ribonucle- 10 mM MnCl2, 50 ng of enzyme.-Exp. 7: 10 mM MnCl2, 32 ng ase H. of enzyme, 0.1 M KCl.-Exp. 8: 1.2 mM MnCl2, 14 ng of enzyme, The solutions were incubated at 350 until the proportion 0.1 M KCl.-Exp. 9: 5.0 mM MnCl2, 70 ng of enzyme.-Exp. 10 is taken from a previous paper (1).-The extent of substrate of radioactivity precipitable by 10% trichloroacetic acid splitting recorded in Exp. 2 in presence of Mg2+ was taken as showed no more drop. This point was reached after an incu- 100%. In all other assays, performed under the optimum condi- bation of 1 hr when about '7 of the radioactivity submitted tions listed above, the degradation values obtained were reduced to enzymic digestion still could be precipitated. At this point by computation to those corresponding to 10 ng of enzyme per % volume of 50% trichloroacetic acid was added to each ves- assay and the results were expressed as % of those obtained in the mixtures were in an ice bath for 30 min and Exp. 2 (Mg2+). "Inactive" means that this cation was inactive sel, kept by itself and that its addition did not affect the activity of an centrifuged in the HB-4 rotor of the Sorvall centrifuge (2000 active metal ion. "Inhibiting" means that the latter was de- X g, 10 min). The sediments were washed by suspension in pressed by the former. 0.5 ml of cold 10% trichloroacetic acid and by centrifugation, carefully drained and taken up, each in 100 gl of 0.66 Mg2+. With all other homopolymer hybrids, the cobalt ion M lithium hydroxide. After the addition of 200 ,g of 0.33 M acts, in fact, as an inhibitor. The metal ion concentrations LiOH to each sample, hydrolysis was performed at 370 for showing optimum activity or 100% inhibition are summa- 18 hr. The procedures for analysis and chromatography rized in Table 2. Manganese would seem to be the "normal" were the same as those described before for the determina- activator of the nuclease; it is the only one permitting the tion of the chain length of oligo(A). hydrolysis of ribo pyrimidine tracts. In Exp. 1, 14.5% of the radioactive riboadenylic acid In order to exclude the possibility that the inhibition by linked covalently to poly(dA) in the copolymer hybrid re- Co2+ was merely an artifact of the analytical method em- mained precipitable after treatment with ribonuclease H; in ployed, by aggregating degradation products, so as to render Exp. 2 the corresponding figure was 14.0%. The balance of them no longer filtrable, a series of experiments was per- the chromatographically separated products of the alkaline formed in which poly(dC)-poly(G), poly(dA)-poly(U), and hydrolysis was (as % of total recovered material): in Exp. 1, (dG)4.poly(C) were treated with the enzyme in presence of adenosine 3',5'-diphosphate, 91; adenosine 2'(3')-monophos- Mn2+. At the end of the incubation, Co2+ was added to an 8 phate, 9. The corresponding values in Exp. 2 were 92% for mM concentration, and then the mixtures were analyzed as pAp and 8% for Ap. This means that after the exhaustive usual. Exactly the same hydrolysis values were found as treatment of hybrid poly[(A)7-(dA)1].poly(dT) with very when Co2+ was omitted. The inhibition, therefore, is not il- large amounts of ribonuclease H the bulk of the resulting lusionary. poly(dA) (82-84%) retained one riboadenylic acid end Enzymic Degradation of Poly[(A)dA~J.Poly(dT) Hy- group, whereas a much smaller proportion (16-18%) still re- brid. The results of two experiments are described here, one mained linked, on the average, to a ribo dinucleotide. with Mg2+, the other with Mn2+ as activator. The experi- Inhibiting Action of SAdenosylmethionine and SAde- mental conditions were as follows. Exp. 1: total volume 1.1 nosylhomocysteine. In our previous publication (1) we de- Downloaded by guest on September 23, 2021 1090 Biochemistry: Stavrianopoulos et al. Proc. Nat. Acad. Sci. USA 73 (1976)

Table 3. Inhibition by S-adenosylmethionine (AdoMet) Table 4. Activity of ribonuclease H after preincubation and S-adenosylhomocysteine (AdoHcy)* with AdoMet or AdoHcy* Temp. of Inhibitor (mM) Substrate Inhi- Inhibitor (mM) Substrate Inhi- Condi- hydrolyzed, bition, preincu- hydrolyzed, bition, tions AdoMet AdoHcy Mrmol % bation AdoMet AdoHcy gmol %

1 a 7.14 0 1 00 6.89 0 2 a 8.0 - 0.34 95 2 00 8.0 6.52 5 3 a - 22.5 1.37 81 3 350 6.75 0 4 b 6.14 0 4 35°. 8.0 0.92 86 5 b 8.0 0.2 97 5 350 22.5 5.95 12 6 b 22.5 1.2 80 6 350 8.0 22.5 3.09 54 7 c 5.6 0 * The substrate was For the preparation of 30 8 c 8.0 0.25 96 poly(dT).poly(A). mM AdoHcy, compare footnote, §. The experimental conditions 9 c 22.5 4.9 12 of preincubation were: total volume, 120 Al; 0.05 M Tris.HCI, pH 10 d 5.6 0 8.0; 25 mM MgCl2; 0.05 M KCl; 400 MLg/ml of bovine serum 11 d 8.0 - 0.19 97 albumin; 120 ng of ribonuclease H; indicated quantities of 12 d 22.5 5.07 9 inhibitor; incubation for 5 min at 00 or 350. After the preincubation, 10 Mu portions of the mixtures (corresponding to 10 ng of * Uniform experimental conditions: incubation for 15 min at 350; enzyme) were added to 110 Ml of the assay mixture, containing total volume, 0.12 ml; 0.05 M Tris.HCl, pH 8.0; 400 Mg/ml of the listed components (but without enzyme and inhibitor), sup- bovine serum albumin; substrate, 83.3 MM with respect to the plemented with poly(dT).poly(A) (83.3 uM with respect to ribonucleotide moiety; indicated quantities of inhibitor. Varying adenylic acid); incubation at 35° for 15 min. experimental conditions: a: poly(dT).poly(A) as substrate; 25 mM MgCl2, 0.05 M KCl, 10 ng of enzyme; b: poly(dC)-poly(G) as substrate; 10 mM MgCl2, 0.05 M KC1, 70 ng of enzyme; c: tor. (The equally great influence of the ionic milieu is left poly(dA).poly(U) as substrate; 1.5 mM MnCl2, 0.1 M KCI, 14 ng out of consideration, since only the optimum conditions are of enzyme; d: (dG)4.poly(C) as substrate; 10 mM MnCl2, 70 ng of specified in this paper.) Tables 1 and 2 illustrate the correla- enzyme. Compare footnote, §, for the inhibition tests with tion between activating metal ion and the composition of the AdoHcy. ribo moiety of the hybrid substrate. Only the hydrolysis of scribed briefly the observation that the activity of ribonucle- hybridized poly(A) is catalyzed by cobalt, that of both pu- ase H is inhibited by S-adenosylmethionine (AdoMet). This rine polyribonucleotides is catalyzed by magnesium, where- effect could have been explained by the assumption that a as manganese is active with all substrates. These findings contaminating protein O-methyltransferase (EC 2.1.1.24), could conceivably lead to a differential method for the spe- which is known to occur in calf thymus (7), could have inac- cific cleavage of a DNA-RNA hybrid. The fi DNA-RNA hy- tivated our enzyme by methylation. This explanation is, brid studied before (4) is cleaved in the presence of each of however, rendered less likely by the finding that S-adenosyl- the three metal activators (Table 1; see also ref. 1), but possi- homocysteine (AdoHcy), although in 3fold concentration, bly into fragments having different end groups according to also acts as an inhibitor of the nuclease, particularly with the metal employed. substrates containing purine ribopolymers.§ A selection of The enzyme appears to require the presence of both a inhibition experiments is presented in Table 3. They show deoxyribo and a ribo strand. Poly(A)-poly(U) is not cleaved that 8 mM AdoMet completely blocks the enzymic degrada- (8), but a systematic study of double-stranded polyribonu- tion of all homopolymer hybrids, whereas 22.5 mM AdoHcy cleotides under a variety of conditions may be interesting. It interferes effectively only with the breakdown of substrates is likely that ribonuclease H must recognize, and attach itself containing poly(A) or poly(G). Other differences between to, a deoxyribo strand, in order to break the complementary these inhibitors are brought out in Table 4. By preincubation polyribonucleotide. This points to the presence in the en- at 350 in the absence of substrate the enzyme is irreversibly zyme of at least two active centers. Asr short a deoxyribo inactivated by AdoMet, whereas AdoHcy has little effect. If component as a tetranucleotide suffices to form hybrids with the contact with the enzyme takes place at 00, however, nei- a polyribo strand susceptible to enzymic cleavage (Table 1). ther AdoMet nor AdoHcy proves inhibitory. What is worthy That this occurs at lower salt concentrations than the attack of note is that the simultaneous pretreatment of the enzyme on an all-poly hybrid may indicate that in the first case it is at 350 with both inhibitors appears to protect the enzyme the conformation of the ribo strand that can impose itself on partially against inactivation by AdoMet. the array of short deoxy chains, producing conditions sufficient for enzymic hydrolysis. The existence, under the con- DISCUSSION ditions of our experiments, of triple strands, of the form 2 ol- The action of ribonuclease H is greatly affected by the com- igo(dM)-poly(N), is most improbable, since these complexes position of the substrate and the nature of the metal activa- require high salt concentrations (9, 10). The hybrid between poly(dA) and poly(U) is interesting: the equimolar hybrid is exists as § Because of the very limited solubility of S-adenosylhomocysteine unstable, and this complex poly(dA).2 poly(U) (10, (AdoHcy) in water, special precautions had to be followed in the 11). It is, in fact, this hybrid which is split by the enzyme preparation of assay mixtures containing this substance. A 30 mM most efficiently in the presence of Mn2+ (Exp. 8 in Table 1), solution of AdoHcy in water was prepared by placing the aqueous possibly owing to the existence of a triple structure. suspension in a completely dust-free tube and heating it to 85° The hybrid between poly(dT) and the block copolymer until all was dissolved. The solution was then brought to pH 7.8 could be regarded as a model of the type of by the addition of 1 M Tris-HCI and stored at 350, under which poly[(Ah-(dA)x] conditions it remained clear for several hours. The final assay product formed in the replication process when a DNA mixture (120 Ml) was made by mixing three portions: 90 A130 mM polymerase is primed by a short RNA chain (12). Ribonucle- AdoHcy, 10 gl enzyme, 20 Ml containing all other components. ase H acting on this hybrid formed degradation products of Downloaded by guest on September 23, 2021 Biochemistry: Stavrianopoulos et al. Proc. Nat. Acad. Sci. USA 73 (1976) 1091 which the bulk (83%) of the poly(deoxyadenylic -acid) Ribonucleases specific for the cleavage of RNA in hybrid moieties retained one riboadenylic acid end group, whereas form are distributed very widely. It is probable that they 17% were linked to a ribodinucleotide.l In this respect the have a significant function in replication or transcription. As calf thymus enzyme resembles the corresponding enzyme concerns the first process, the enzyme may be operative in from E. coil (13). removing the RNA primer portion of a newly synthesized This finding is of interest, since it predicts that under bio- DNA chain (15). Once this is accomplished, the complemen- logical conditions a polymer of the type p(rM)m-(dN)n, tary DNA segment is left unpaired and available either for a which is bonded to a complementary all-deoxy strand, will repair process by DNA polymerase and ligase or as an initia- be attacked by ribonuclease H in such a manner as to retain tor site for RNA polymerase B, which is known to prefer sin-

the grouping ... prM-dNp .... The remaining mono- or di- gle-stranded DNA (16). If the transcription by this enzyme, ribonucleotide residue would then have to be removed by bound initially to an unpaired DNA region produced as de- other mechanisms unless it is retained in the completed scribed here or in another manner, continues into the dou- DNA molecule. ble-stranded portion of the DNA, the stretch of RNA tran- Preliminary experiments, not detailed in this paper, have scribed from the latter will be extruded but remain at- made it likely that also in the counterpart of the hybrid dis- tached, as an initial hybrid, to the initiator site. This hybrid cussed in the preceding paragraph, namely, in a hybrid be- segment again is a substrate for ribonuclease H, which thus tween poly(dT) and p(dA)8-(A)2, one of the two ribonucleo- would effect the release of the nonhybridized part of the tides is removed by the enzyme, leaving, on the average, one RNA transcript. In this manner, the participation of ribonu- riboadenylic acid residue linked 5' -- 3' to the octadeoxyr- clease H in the production, for instance, of heterogeneous bonucleotide. nuclear RNA could be envisioned. Turning to the inhibition experiments described here, it This work was supported by U.S. Public Health Service Grant no. will be noticed that cobalt ions, which activate the enzymic CA-12210. cleavage of poly(A) hybrids, inhibit that of all other homopolymer substrates (Table 2), most probably owing to the ef- 1. Stavrianopoulos, J. G. & Chargaff, E. (1973) Proc. Nat. Acad. fect of the metal on the configuration of the substrates rath- Sci. USA 70, 1959-1963. er than of the enzyme. A more specific inhibitory effect, 2. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) namely, of S-adenosylmethionine, was mentioned before (1). Proc. Nat. Acad. Sci. USA 69,1781-1785. 3. Kato, K., J. M., Houts, E. & F. It is shown in more detail in Tables 3 and 4. This substance Goncalves, G. Bollum, J. (1967) Biol. Chem. 242,2780-2789. inactivates enzyme on at J. the irreversibly preincubation 350, 4. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) but not at 00. The assumption that inactivation may be due Proc. Nat. Acad. Sci. USA 69,2609-2613. to enzymic methylation would seem to be contradicted by 5. Farber, F. E. & Chargaff, E. (1967) Eur. J. Biochem. 2, 433- the observation that S-adenosylhomocysteine in a higher 441. concentration also acts as an inhibitor, although not irrevers- 6. Uhlenbeck, O., Harrison, R. & Doty, P. (1968) in Molecular ibly. Partial protection from irreversible inactivation is af- Associations in Biology, ed. Pullman, B. (Academic Press, forded by simultaneous treatment with both nucleoside de- New York and London), pp. 107-114. rivatives, which may mean that both inhibitors compete for 7. Paik, W. K. & Kim, S. (1968) J. Biol. Chem. 243,2108-2114. the same site on the enzyme. Since relatively high concen- 8. Hausen, P. & Stein, H. (1970) Eur. J. Biochem. 14,278-283. 9. trations (6-8 mM) of S-adenosylmethionine are required for Rich, A. (1960) Proc. Nat. Acad. Sci. USA 46,1044-1053. 10. Riley, M., Maling, B. & Chamberlin, M. J. (1966) J. Mol. Biol. the inactivation of ribonuclease H-nearly the thousandfold 20,359-389. amount of what is needed for enzymic methyl transfer 11. Felsenfeld, G. & Rich, A. (1957) Biochim. Biophys. Acta 28, (14)-even the nonenzymic methylation of the enzyme 457-468. could be envisioned. 12. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1971) Proc. Nat. Acad. Sc. USA 68, 2207-2211. 13. I If an oligoribonucleotide is not stabilized by covalent 3' - 5' link- Leis, J. P., Berkower, I. & Hurwitz, J. (1973) Proc. Nat. Acad. age to a polydeoxyribonucleotide, as in this experiment, ribonu- Sci. USA 70,466-470. clease H produces fewer breaks. For instance, a hybrid between 14. Jamaluddin, M., Kim, S. & Paik, W. K. (1975) Biochemistry poly(dT) and (A)7 yielded, on treatment with large amounts of 14,694-698. the enzyme, fragments of an average length of 3, presumably be- 15. Keller, W. (1972) Proc. Nat. Acad. Sci. USA 69,1560-1564. cause even one break resulted in pieces that were too short to 16. Chambon, P. (1974) in The Enzymes, ed. Boyer, P. D., (Aca- maintain the stable hybrid structure that is requisite for enzymic demic Press, New York and London), 3rd ed., Vol. X, pp. cleavage. 261-331. Downloaded by guest on September 23, 2021