Proc. Nat. Acad. Sc. USA
Vol. 73, No. 4, pp.
Biochemistry
1087-1091!, April 1976
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 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, Cot+ only of poly(A) hybrids. A 1:1 hybrid of phage fi 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 dA)J. 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 Sadenosylmethionine at 350, but not at 0°. 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. We continue our study of ribonuclease H (hybrid nuclease; RNA-DNA hybrid ribonucleotidohydrolase; EC 3.1.4.34) of calf thymus (1) by directing our attention to problems of substrate specificity, the influence of different metals on the course of substrate hydrolysis, and the specific inhibition of the enzyme.
MATERIALS In addition to materials listed before (1) the following preparations were employed. Ribonucleoside diphosphates and Sadenosylhomocysteine came from Sigma, S-adenosylmethionine sulfate from Boehringer, oligodeoxynucleotides (di, tetra, octa) from Collaborative Research, Inc. Hydroxylapatite was used as Bio-Gel HTP (Bio-Rad). Most enzymes em-
ployed, including the highly purified thymus ribonuclease H, have been described before (1). Polynucleotide phosphorylase (EC 2.7.7.8) was obtained as by-product during the isolation of RNA polymerase from Escherichia coil B (EC 2.7.7.6). The latter enzyme preparation was free of polynucleotide phosphorylase and of ribonuclease. The DNA polymerase (EC 2.7.7.7) of chicken embryo (2) had been stored 2 Abbreviations: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine. * Present address to which reprint requests should be sent: Cell Chemistry Laboratory, The Roosevelt Hospital, 428 West 59th St., New York, N.Y. 10019. 1087
years. Pancreatic deoxyribonuclease I, free of ribonuclease (EC 3.1.4.5), was a Worthington product. SUBSTRATES Homopolymers. The polydeoxynucleotides were synthesized with the aid of the terminal deoxynucleotidyl transferase of calf thymus (3), in the presence of Mg2+ ions for poly(dA) and of Co2+ ions for poly(dC) and poly(dT). The phage fI DNA-RNA hybrid (1:1) has been described before (4). The 3H-labeled ribopolymers poly(U) and poly(C) were prepared and purified by a modification of a previous procedure (5). The (dA)-(U), (dT)-(A), and (dG)-(C) hybrids were made by mixing equimolar quantities of the complementary homopolymers in 0.05 M Tris-HCI (pH 8.0); they were stored at -20°. For the (dC)-(G) hybrids, the components were kept at 780 for 4 hr, then cooled overnight to 450, and the hybrids were stored at 00. Radioactive poly(A) was made by the action of RNA polymerase on poly(dT) in the presence of [3H]ATP (2600 cpm/nmol) or [14C]ATP (7000 cpm/nmol) as described (1). Radioactive poly(G) was made on a poly(dC) template. The 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 poly(dC), and 7 units/ml of RNA polymerase, in 0.05 M Tris-HCI of pH 8.5 (at 350). On incubation at 350 under argon, polymerization is very fast initially, but progresses slowly and nearly comes to a stop at 20 hr. The yield of poly(G) varied widely, between 25 and 82% of the poly(dC) template employed. After addition of 14 ml of ethanol and storage overnight at 40, the precipitate was collected by centrifugation (10,000 X g, 10 min), and its solution in 2 ml of the Tris.HCI buffer was dialyzed for 3 days against 0.05 M KCI to remove traces of GTP. Poly(G) was obtained by treating the poly(G)-poly(dC) hybrid with deoxyribonuclease in 0.02 M Tris-HCI (pH 7.5) and 0.001 M MnCI2. After dialysis for 24 hr against the same buffer at room temperature, the Mn salt of poly(G) was collected by centrifugation and taken up in 0.001 M EDTA. Oligoadenylate. Radioactive oligo(A) with a free terminal 3' hydroxyl was prepared by partial hydrolysis of the hybrid of poly(dT) with poly(['4C]A) (7000 cpm/nmol), described before, with the aid of ribonuclease H. The incubation mixture (8 ml) consisted of 25 mM MgCl2, 0.05 mM KCI, 250 '4g/ml of bovine serum albumin, a quantity of the hybrid poly(dT)-poly(A) corresponding to 187.5 nmol/ml of adenylic acid, 125 units/ml of ribonuclease H (1), with the enzyme activity determined in the presence of Mg2+, in 0.05 M Tris-HCI of pH 8.0. The incubation mixture without the enzyme was brought to 350 and the reaction was then started by the addition of the enzyme. After 15 min at 350, the
1088
Biochemistry: Stavrianopoulos et al.
mixture was adjusted to pH 2.0-2.5 with 1 M HCl, thus inactivating the enzyme, and left at this pH for 10 min. Following neutralization with 1 M Tris base and adjustment to 37.5 mM EDTA, the solution was applied to a column of 2 ml of hydroxylapatite equilibrated with 0.15 M KCL. Elution was performed with potassium phosphate buffer of pH 6.8: 0.025 M for the oligonucleotides and 0.3 M for the unhydrolyzed hybrid. The 0.025 M eluate was placed on a column of 0.5 ml of DEAE-cellulose equilibrated with 0.05 M Tris.HCl of pH 8.0.t The small oligonucleotides (n < 5) were eluted with 8 ml of 0.2 M KC1 (flow rate 4 ml/hr) and the oligonucleotides of an average length of n = 7.2 were eluted with 0.4 M KCl at the same flow rate, the first 300 Mtl of eluate being discarded. Four fractions each of 0.5 ml were collected, of which the first three contained 4.561 X 106 cpm or 43.4% of the poly([14C]A) submitted to enzymic hydrolysis; the fourth eluate fraction corresponded to only 3% of the original radioactivity and was discarded. The proportion of radioactivity precipitable by trichloroacetic acid was determined 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, 14.3%. The average length of the oligo(A) preparation was estimated by alkaline hydrolysis (0.66 M LiOH, 370, 18 hr) and determination of adenosine, 2'(3')-adenylic acid, and adenosine 3',5'-diphosphate (paper chromatography in 1propanol/concentrated ammonia/water, 11:2:7, vol/vol/ vol). Since the length was found as 7.2 (based on pAp) or 7.5 (based on adenosine), this preparation is designated as (A)7.* Hybrid of Poly[(AHdA~J and Poly(dT). This copolymer hybrid was made by the action of the DNA polymerase of chicken embryo (2) obi the hybrid of poly(dT) with the 14Clabeled ribo oligomer p(A)7 in the presence of dATP: The incubation mixture (12 ml) consisted of 1.25 mM MnCl2, 83 mM KC1, 1.7 mM dithiothreitol, 400,uM dATP, 500,MM poly(dT), 50 MuM (A)7, 33 units/ml of DNA polymerase, and 250 ug/ml of bovine serum albumin, in 0.05 M Tris.HCl of pH 8.0. In order to facilitate a uniform distribution of the oligoadenylate on the poly(dT) present in a 10-fold excess, the following procedure was employed. A solution of the (Ah and the poly(dT) in 7 ml of 0.116 M KCl was kept at 700 for 10 min and then slowly brought to room temperature, the rest of the additives (minus enzyme) in 4.9 ml of buffer was admixed in an argon atmosphere, and the mixture was cooled to 00. Then the enzyme was added and incubation was performed at 200 for 12 hr (compare Table 1 in ref. 4). The radioactivity and its precipitability with trichloroacetic acid at zero time and at the end of the incubation were measured in duplicate in 50 gl portions. Per 50 Ml portion there were 17,181 cpm, of which 1226 (7.1%) were precipitable at zero time and 14,275 (83.1%) were precipitable at the end of the reaction. In order to remove unincorporated oligonucleotides, the incubation mixture was adjusted to 2.0 mM EDTA and to pH 7.0 by M Tris.HCI and then placed on a column of 1.5 ml of hydroxylapatite equilibrated with 0.15 M KCI. The unpolymerized oligonucleotides were eluted with 0.05 M potassium phosphate of pH 6.8, followed by the elution with t
Microcolumns are prepared conveniently by using the polypropylene tip of a 1 ml Eppendorf automatic pipette closed with a small plug of glass wool. The average chain length of these products is, of course, not entirely the same in different experiments. For instance, in an assay in which poly([3H]A)-poly(dT) was treated with ribonuclease H by a somewhat different procedure, the resulting oligo(A) had an average chain length of 8.9.
Proc. Nat. Acad. Sci. USA 73 (1976) 0.5 M potassium phosphate (pH 6.8) of the hybrid poly(dT)-
poly[(A)7-(dA),]. The balance of radioactivity (as nmol of [14C]adenylic acid) was: placed on a column, 489.4; eluted as oligonucleotides, 88.3 (18.0%); eluted as polymer hybrid, 314.9 (64.3%). A portion, 17.6% of the radioactivity, was not
eluted, presumably held back on the column as an enzymepolynucleotide complex. The 0.5 M potassium phosphate eluate of the polymer hybrid was freed of phosphate by being passed through a Sephadex G-25 column equilibrated with 0.1 M KCl or 0.2 M ammonium sulfate. This hybrid was completely resistant to pancreatic ribonuclease, as shown by the amount of radioactivity precipitable with 10% trichloroacetic acid at zero time and after enzyme treatment. In parallel experiments (in each: 55 gl solution, 0.1 M KCI, 0.01 M EDTA, pH 7.5, 15,000 cpm, 100 Mug of ribonuclease, 350, 30 min), the radioactivity precipitable at zero time was 14,930 and 15,050 cpm, and after ribonuclease treatment, 15,070 and 14,540 cpm. RESULTS Factors Influencing the Action of Ribonuclease H on
Homopolymer Hybrids. The base composition of the ribo strand, the length of the deoxyribo strand, the nature and the concentration of the metal activator, and also the salt concentration of the medium (left out of consideration here), all these determine, though to a varying degree, the course and the outcome of the enzymic action. Base Composition of Ribo Strand. Poly(dT)-poly(A), in the presence of Mg2+, is the best substrate, resembling the fi DNA-RNA hybrid (1:1) discussed previously (1) in its hydrolysis requirements. Poly(dC)-poly(G) is attacked much more slowly, requiring four times as long for the same degree of splitting as poly(dT)-poly(A) in the presence of Mn2+. The results summarized in Table 1 illustrate the variety of conditions governing the enzymic hydrolysis of the homopolymer hybrids under study. The fl hybrid mentioned before is included for comparison. The normalization of all results, required for comparison, is also explained in this table. Length of Deoxyribo Strand. As is also shown in Table 1, the degradation velocity rises with the increasing length of the deoxyribo strand. It is, however, remarkable that even deoxyribonucleotides as short as tetra offer sufficient support for the ribo strand to be attacked by the hybrid-specific enzyme, since the temperature of the enzyme reaction, 350, presumably lies above the melting temperatures of the respective double structures (6). It would not be surprising if the stability of the hydrogen-bonded alignment of the two strands were, in fact, increased by the enzyme as it proceeds to degrade the hybrid. The study of the effect of deoxyribo tetranucleotides, of which all four representatives could be examined (Exps. 1, 3, 6, and 9 in Table 1), offers an opportunity of comparing the influence of the particular base on the hydrolysis of the complementary ribo strand. The efficiency decreases in the following order: (dA)4.poly(U); (dG)4. poly(C); (dC)4.poly(G); (dT)4.poly(A), when Mn2+ is the activator. Pyrimidine ribo strands are broken more readily than purine strands. The only deoxyribo dinucleotide available, d(pApA), did not support the enzymic degradation of the complementary polyribo strand (Exp. 5). Divalent Metal Ions. The differential effects are somewhat baffling. Mn 2+ activates the attack on all hybrid combinations; Mg2+, only on those containing purine ribo strands; Co2+, only on poly(A) hybrids, but less well than
Biochemistry: Stavrianopoulos et al.
Proc. Nat. Acad. Sci. USA 73 (1976)
Table 1. Degradation efficiency of different hybrid substrates by ribonuclease H*
Components of equimolar hybrid Ribo Deoxyribo 1 2 3 4 5 6 7 8 9 10
(dT)4 Poly(dT)
(dC)4 Poly(dC) (dA)2
(dA)4
(dA), Poly(dA)
(dG)4 fl DNA
Poly(A) Poly(A) Poly(G) Poly(G) Poly(U) Poly(U) Poly(U) Poly(U) Poly(C) RNA tran-
script
Mg2+ 25 100 7.8 12.5 Inact. Inact. Inact. Inact. Inact.
66.5
% degradation Mn2+ C02+ 6.3 40 6.4 10.4
2.1 70 Inhib. Inhib. Inact. Inhib. Inhib.
Inact. 17.5 28.2
Inhib.
62.5
Inhib.
12.5
61.7
54.3
Each assay sample (120 ul) consisted of 0.05 M Tris-HCl (pH 8.0), of hybrid substrate corresponding to 83.3 MM with respect to the ribo moiety, of 0.4 mg/ml of bovine serum albumin, and of the additions specified below for each experiment. The indicated 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 mM MnCl2, 135 ng of enzyme, 0.1 M (NH4)2S04; 2.5 mM CoC12, 420 ng of enzyme, 0.2 M KCl.-Exp. 2: 25 mM MgCl2, 10 ng of enzyme, 0.05 M KQl; 1.5 mM MnCl2, 20 ng of enzyme, 0.2 M (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 enzyme.-Exp. 4: 10 mM MgCl2, 70 ng of enzyme, 0.05 M KQl; 0.25 mM MnCl2, 80 ng of enzyme, 0.15 M KCl.-Exps. 5 and 6: 10 mM MnCl2, 50 ng of enzyme.-Exp. 7: 10 mM MnCl2, 32 ng of enzyme, 0.1 M KCl.-Exp. 8: 1.2 mM MnCl2, 14 ng of enzyme, 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 splitting recorded in Exp. 2 in presence of Mg2+ was taken as 100%. In all other assays, performed under the optimum conditions listed above, the degradation values obtained were reduced by computation to those corresponding to 10 ng of enzyme per assay and the results were expressed as % of those obtained in Exp. 2 (Mg2+). "Inactive" means that this cation was inactive by itself and that its addition did not affect the activity of an active metal ion. "Inhibiting" means that the latter was depressed by the former.
*
Mg2+. With all other homopolymer hybrids, the cobalt ion fact, as an inhibitor. The metal ion concentrations showing optimum activity or 100% inhibition are summarized in Table 2. Manganese would seem to be the "normal" activator of the nuclease; it is the only one permitting the hydrolysis of ribo pyrimidine tracts. In order to exclude the possibility that the inhibition by Co2+ was merely an artifact of the analytical method employed, by aggregating degradation products, so as to render them no longer filtrable, a series of experiments was performed in which poly(dC)-poly(G), poly(dA)-poly(U), and (dG)4.poly(C) were treated with the enzyme in presence of Mn2+. At the end of the incubation, Co2+ was added to an 8 mM concentration, and then the mixtures were analyzed as usual. Exactly the same hydrolysis values were found as when Co2+ was omitted. The inhibition, therefore, is not illusionary. Enzymic Degradation of Poly[(A)dA~J.Poly(dT) Hybrid. The results of two experiments are described here, one with Mg2+, the other with Mn2+ as activator. The experimental conditions were as follows. Exp. 1: total volume 1.1 acts, in
Table 2. Effects of metal ions on ribonuclease H* Metal ion (mM)
Effect of metal on
1089
Components of equimolar hybrid Deoxyribo
Ribo
(dT)4 Poly(dT) (dC)4 Poly(dC) (dA)4
Poly(A) Poly(A) Poly(G)
(dA)s Poly(dA) (dG)4 fl DNA
Poly(G) Poly(U) Poly(U) Poly(U) Poly(C)
Optimum activity C0o2+ Mn2+ Mg2+ 1,5 1.5 0.4 0.4 10 10
10 25 12 10 Inact.
1.5 7.5
Inact. Inact. Inact.
1.5
25
100% Inhibition
Co2+
2.5 20 8 8 1.5 1.5
1.5 1
RNA tran-
script
20
*The experimental conditions were those indicated in Table 1, with the enzyme and salt concentrations specified there. The concentrations of metal activators or inhibitors varied, however, over the range shown here.
ml; 0.05 M Tris-HUl, pH 8.0; 0.025 M MgC12; 0.05 M KCl; 500 .g of bovine serum albumin; copolymer hybrid corresponding to 51.4 nmol of riboadenylic acid; and 500 units of ribonuclease H.-Exp. 2: total volume 1.3 ml; 0.05 M TrisHC1, pH 8.0; 2.0 mM MnCl2; 0.2 M (NH42S04; 500 ,ug of bovine serum albumin; copolymer hybrid corresponding to 53.3 nmol of riboadenylic acid; and 500 units of ribonuclease H. The solutions were incubated at 350 until the proportion of radioactivity precipitable by 10% trichloroacetic acid showed no more drop. This point was reached after an incubation of 1 hr when about '7 of the radioactivity submitted to enzymic digestion still could be precipitated. At this point % volume of 50% trichloroacetic acid was added to each vessel, the mixtures were kept in an ice bath for 30 min and centrifuged in the HB-4 rotor of the Sorvall centrifuge (2000 X g, 10 min). The sediments were washed by suspension in 0.5 ml of cold 10% trichloroacetic acid and by centrifugation, carefully drained and taken up, each in 100 gl of 0.66 M lithium hydroxide. After the addition of 200 ,g of 0.33 M LiOH to each sample, hydrolysis was performed at 370 for 18 hr. The procedures for analysis and chromatography were the same as those described before for the determination of the chain length of oligo(A). In Exp. 1, 14.5% of the radioactive riboadenylic acid
linked covalently to poly(dA) in the copolymer hybrid remained precipitable after treatment with ribonuclease H; in Exp. 2 the corresponding figure was 14.0%. The balance of the chromatographically separated products of the alkaline hydrolysis was (as % of total recovered material): in Exp. 1, adenosine 3',5'-diphosphate, 91; adenosine 2'(3')-monophosphate, 9. The corresponding values in Exp. 2 were 92% for pAp and 8% for Ap. This means that after the exhaustive treatment of hybrid poly[(A)7-(dA)1].poly(dT) with very large amounts of ribonuclease H the bulk of the resulting poly(dA) (82-84%) retained one riboadenylic acid end group, whereas a much smaller proportion (16-18%) still remained linked, on the average, to a ribo dinucleotide. Inhibiting Action of SAdenosylmethionine and SAdenosylhomocysteine. In our previous publication (1) we de-
1090
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Stavrianopoulos et al.
Table 4. Activity of ribonuclease H after preincubation with AdoMet or AdoHcy*
Table 3. Inhibition by S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy)* Substrate
Inhi-
0 95 81 0 97 80 0
-
7.14 0.34 1.37 6.14 0.2 1.2 5.6 0.25 4.9 5.6 0.19
22.5
5.07
Inhibitor (mM)
Conditions AdoMet 1 2 3 4 5 6 7 8 9 10 11 12 *
a a a
b b b
AdoHcy
8.0
-
-
22.5
8.0 22.5
c c
8.0
22.5
c
d d
d
8.0
96
12 0
97 9
Uniform experimental conditions: incubation for 15 min at 350; total volume, 0.12 ml; 0.05 M Tris.HCl, pH 8.0; 400 Mg/ml of bovine serum albumin; substrate, 83.3 MM with respect to the ribonucleotide moiety; indicated quantities of inhibitor. Varying 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: poly(dA).poly(U) as substrate; 1.5 mM MnCl2, 0.1 M KCI, 14 ng of enzyme; d: (dG)4.poly(C) as substrate; 10 mM MnCl2, 70 ng of enzyme. Compare footnote, §, for the inhibition tests with AdoHcy.
scribed briefly the observation that the activity of ribonuclease H is inhibited by S-adenosylmethionine (AdoMet). This effect could have been explained by the assumption that a contaminating protein O-methyltransferase (EC 2.1.1.24), which is known to occur in calf thymus (7), could have inactivated our enzyme by methylation. This explanation is, however, rendered less likely by the finding that S-adenosylhomocysteine (AdoHcy), although in 3fold concentration, also acts as an inhibitor of the nuclease, particularly with substrates containing purine ribopolymers.§ A selection of inhibition experiments is presented in Table 3. They show that 8 mM AdoMet completely blocks the enzymic degradation of all homopolymer hybrids, whereas 22.5 mM AdoHcy interferes effectively only with the breakdown of substrates containing poly(A) or poly(G). Other differences between these inhibitors are brought out in Table 4. By preincubation at 350 in the absence of substrate the enzyme is irreversibly inactivated by AdoMet, whereas AdoHcy has little effect. If the contact with the enzyme takes place at 00, however, neither AdoMet nor AdoHcy proves inhibitory. What is worthy of note is that the simultaneous pretreatment of the enzyme at 350 with both inhibitors appears to protect the enzyme partially against inactivation by AdoMet. DISCUSSION
The action of ribonuclease H is greatly affected by the composition of the substrate and the nature of the metal activa§
InhiInhibitor (mM) Substrate Temp. of hydrolyzed, bition, preincugmol % bation AdoMet AdoHcy
hydrolyzed, bition, Mrmol %
Because of the very limited solubility of S-adenosylhomocysteine (AdoHcy) in water, special precautions had to be followed in the preparation of assay mixtures containing this substance. A 30 mM solution of AdoHcy in water was prepared by placing the aqueous suspension in a completely dust-free tube and heating it to 85° until all was dissolved. The solution was then brought to pH 7.8 by the addition of 1 M Tris-HCI and stored at 350, under which conditions it remained clear for several hours. The final assay mixture (120 Ml) was made by mixing three portions: 90 A130 mM AdoHcy, 10 gl enzyme, 20 Ml containing all other components.
1 2 3 4 5 6 *
00 00
8.0
350 35°. 350
8.0
350
8.0
22.5 22.5
6.89 6.52 6.75 0.92 5.95 3.09
0 5 0 86 12 54
The substrate was poly(dT).poly(A). For the preparation of 30 mM AdoHcy, compare footnote, §. The experimental conditions of preincubation were: total volume, 120 Al; 0.05 M Tris.HCI, pH 8.0; 25 mM MgCl2; 0.05 M KCl; 400 MLg/ml of bovine serum albumin; 120 ng of ribonuclease H; indicated quantities of inhibitor; incubation for 5 min at 00 or 350. After the preincubation, 10 Mu portions of the mixtures (corresponding to 10 ng of enzyme) were added to 110 Ml of the assay mixture, containing the listed components (but without enzyme and inhibitor), supplemented with poly(dT).poly(A) (83.3 uM with respect to adenylic acid); incubation at 35° for 15 min.
tor. (The equally great influence of the ionic milieu is left out of consideration, since only the optimum conditions are specified in this paper.) Tables 1 and 2 illustrate the correla-
tion between activating metal ion and the composition of the ribo moiety of the hybrid substrate. Only the hydrolysis of hybridized poly(A) is catalyzed by cobalt, that of both purine polyribonucleotides is catalyzed by magnesium, whereas manganese is active with all substrates. These findings could conceivably lead to a differential method for the specific cleavage of a DNA-RNA hybrid. The fi DNA-RNA hybrid studied before (4) is cleaved in the presence of each of the three metal activators (Table 1; see also ref. 1), but possibly into fragments having different end groups according to the metal employed. The enzyme appears to require the presence of both a deoxyribo and a ribo strand. Poly(A)-poly(U) is not cleaved (8), but a systematic study of double-stranded polyribonucleotides under a variety of conditions may be interesting. It is likely that ribonuclease H must recognize, and attach itself to, a deoxyribo strand, in order to break the complementary polyribonucleotide. This points to the presence in the enzyme of at least two active centers. Asr short a deoxyribo component as a tetranucleotide suffices to form hybrids with a polyribo strand susceptible to enzymic cleavage (Table 1). That this occurs at lower salt concentrations than the attack on an all-poly hybrid may indicate that in the first case it is the conformation of the ribo strand that can impose itself on the array of short deoxy chains, producing conditions sufficient for enzymic hydrolysis. The existence, under the conditions of our experiments, of triple strands, of the form 2 oligo(dM)-poly(N), is most improbable, since these complexes require high salt concentrations (9, 10). The hybrid between poly(dA) and poly(U) is interesting: the equimolar hybrid is unstable, and this complex exists as poly(dA).2 poly(U) (10, 11). It is, in fact, this hybrid which is split by the enzyme most efficiently in the presence of Mn2+ (Exp. 8 in Table 1), possibly owing to the existence of a triple structure. The hybrid between poly(dT) and the block copolymer poly[(Ah-(dA)x] could be regarded as a model of the type of product formed in the replication process when a DNA polymerase is primed by a short RNA chain (12). Ribonuclease H acting on this hybrid formed degradation products of
Biochemistry: Stavrianopoulos et al.
Proc. Nat. Acad. Sci. USA 73 (1976)
which the bulk (83%) of the poly(deoxyadenylic -acid) moieties retained one riboadenylic acid end group, whereas 17% were linked to a ribodinucleotide.l In this respect the calf thymus enzyme resembles the corresponding enzyme from E. coil (13). This finding is of interest, since it predicts that under biological conditions a polymer of the type p(rM)m-(dN)n, which is bonded to a complementary all-deoxy strand, will be attacked by ribonuclease H in such a manner as to retain The remaining mono- or dithe grouping prM-dNp ribonucleotide residue would then have to be removed by other mechanisms unless it is retained in the completed DNA molecule. Preliminary experiments, not detailed in this paper, have made it likely that also in the counterpart of the hybrid discussed in the preceding paragraph, namely, in a hybrid between poly(dT) and p(dA)8-(A)2, one of the two ribonucleotides is removed by the enzyme, leaving, on the average, one riboadenylic acid residue linked 5' 3' to the octadeoxyrbonucleotide. Turning to the inhibition experiments described here, it will be noticed that cobalt ions, which activate the enzymic cleavage of poly(A) hybrids, inhibit that of all other homopolymer substrates (Table 2), most probably owing to the effect of the metal on the configuration of the substrates rather than of the enzyme. A more specific inhibitory effect, namely, of S-adenosylmethionine, was mentioned before (1). It is shown in more detail in Tables 3 and 4. This substance inactivates the enzyme irreversibly on preincubation at 350, but not at 00. The assumption that inactivation may be due to enzymic methylation would seem to be contradicted by the observation that S-adenosylhomocysteine in a higher concentration also acts as an inhibitor, although not irreversibly. Partial protection from irreversible inactivation is afforded by simultaneous treatment with both nucleoside derivatives, which may mean that both inhibitors compete for the same site on the enzyme. Since relatively high concentrations (6-8 mM) of S-adenosylmethionine are required for the inactivation of ribonuclease H-nearly the thousandfold amount of what is needed for enzymic methyl transfer (14)-even the nonenzymic methylation of the enzyme could be envisioned. ...
....
--
I
If an oligoribonucleotide is not stabilized by covalent 3' 5' linkage to a polydeoxyribonucleotide, as in this experiment, ribonuclease H produces fewer breaks. For instance, a hybrid between poly(dT) and (A)7 yielded, on treatment with large amounts of the enzyme, fragments of an average length of 3, presumably because even one break resulted in pieces that were too short to maintain the stable hybrid structure that is requisite for enzymic cleavage. -
1091
Ribonucleases specific for the cleavage of RNA in hybrid form are distributed very widely. It is probable that they have a significant function in replication or transcription. As concerns the first process, the enzyme may be operative in removing the RNA primer portion of a newly synthesized DNA chain (15). Once this is accomplished, the complementary DNA segment is left unpaired and available either for a repair process by DNA polymerase and ligase or as an initiator site for RNA polymerase B, which is known to prefer single-stranded DNA (16). If the transcription by this enzyme, bound initially to an unpaired DNA region produced as described here or in another manner, continues into the double-stranded portion of the DNA, the stretch of RNA transcribed from the latter will be extruded but remain attached, as an initial hybrid, to the initiator site. This hybrid segment again is a substrate for ribonuclease H, which thus would effect the release of the nonhybridized part of the RNA transcript. In this manner, the participation of ribonuclease H in the production, for instance, of heterogeneous nuclear RNA could be envisioned. This work was supported by U.S. Public Health Service Grant no. CA-12210. 1. Stavrianopoulos, J. G. & Chargaff, E. (1973) Proc. Nat. Acad. Sci. USA 70, 1959-1963. 2. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) Proc. Nat. Acad. Sci. USA 69,1781-1785. 3. Kato, K., Goncalves, J. M., Houts, G. E. & Bollum, F. J. (1967)
J. Biol. Chem. 242,2780-2789. 4. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) Proc. Nat. Acad. Sci. USA 69,2609-2613. 5. Farber, F. E. & Chargaff, E. (1967) Eur. J. Biochem. 2, 433441. 6. Uhlenbeck, O., Harrison, R. & Doty, P. (1968) in Molecular Associations in Biology, ed. Pullman, B. (Academic Press, New York and London), pp. 107-114. 7. Paik, W. K. & Kim, S. (1968) J. Biol. Chem. 243,2108-2114. 8. Hausen, P. & Stein, H. (1970) Eur. J. Biochem. 14,278-283. 9. Rich, A. (1960) Proc. Nat. Acad. Sci. USA 46,1044-1053. 10. Riley, M., Maling, B. & Chamberlin, M. J. (1966) J. Mol. Biol.
20,359-389. 11. Felsenfeld, G. & Rich, A. (1957) Biochim. Biophys. Acta 28, 457-468. 12. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1971) Proc. Nat. Acad. Sc. USA 68, 2207-2211. 13. Leis, J. P., Berkower, I. & Hurwitz, J. (1973) Proc. Nat. Acad. Sci. USA 70,466-470. 14. Jamaluddin, M., Kim, S. & Paik, W. K. (1975) Biochemistry 14,694-698. 15. Keller, W. (1972) Proc. Nat. Acad. Sci. USA 69,1560-1564. 16. Chambon, P. (1974) in The Enzymes, ed. Boyer, P. D., (Academic Press, New York and London), 3rd ed., Vol. X, pp. 261-331.
Vol. 73, No. 4, pp.
Biochemistry
1087-1091!, April 1976
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 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, Cot+ only of poly(A) hybrids. A 1:1 hybrid of phage fi 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 dA)J. 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 Sadenosylmethionine at 350, but not at 0°. 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. We continue our study of ribonuclease H (hybrid nuclease; RNA-DNA hybrid ribonucleotidohydrolase; EC 3.1.4.34) of calf thymus (1) by directing our attention to problems of substrate specificity, the influence of different metals on the course of substrate hydrolysis, and the specific inhibition of the enzyme.
MATERIALS In addition to materials listed before (1) the following preparations were employed. Ribonucleoside diphosphates and Sadenosylhomocysteine came from Sigma, S-adenosylmethionine sulfate from Boehringer, oligodeoxynucleotides (di, tetra, octa) from Collaborative Research, Inc. Hydroxylapatite was used as Bio-Gel HTP (Bio-Rad). Most enzymes em-
ployed, including the highly purified thymus ribonuclease H, have been described before (1). Polynucleotide phosphorylase (EC 2.7.7.8) was obtained as by-product during the isolation of RNA polymerase from Escherichia coil B (EC 2.7.7.6). The latter enzyme preparation was free of polynucleotide phosphorylase and of ribonuclease. The DNA polymerase (EC 2.7.7.7) of chicken embryo (2) had been stored 2 Abbreviations: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine. * Present address to which reprint requests should be sent: Cell Chemistry Laboratory, The Roosevelt Hospital, 428 West 59th St., New York, N.Y. 10019. 1087
years. Pancreatic deoxyribonuclease I, free of ribonuclease (EC 3.1.4.5), was a Worthington product. SUBSTRATES Homopolymers. The polydeoxynucleotides were synthesized with the aid of the terminal deoxynucleotidyl transferase of calf thymus (3), in the presence of Mg2+ ions for poly(dA) and of Co2+ ions for poly(dC) and poly(dT). The phage fI DNA-RNA hybrid (1:1) has been described before (4). The 3H-labeled ribopolymers poly(U) and poly(C) were prepared and purified by a modification of a previous procedure (5). The (dA)-(U), (dT)-(A), and (dG)-(C) hybrids were made by mixing equimolar quantities of the complementary homopolymers in 0.05 M Tris-HCI (pH 8.0); they were stored at -20°. For the (dC)-(G) hybrids, the components were kept at 780 for 4 hr, then cooled overnight to 450, and the hybrids were stored at 00. Radioactive poly(A) was made by the action of RNA polymerase on poly(dT) in the presence of [3H]ATP (2600 cpm/nmol) or [14C]ATP (7000 cpm/nmol) as described (1). Radioactive poly(G) was made on a poly(dC) template. The 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 poly(dC), and 7 units/ml of RNA polymerase, in 0.05 M Tris-HCI of pH 8.5 (at 350). On incubation at 350 under argon, polymerization is very fast initially, but progresses slowly and nearly comes to a stop at 20 hr. The yield of poly(G) varied widely, between 25 and 82% of the poly(dC) template employed. After addition of 14 ml of ethanol and storage overnight at 40, the precipitate was collected by centrifugation (10,000 X g, 10 min), and its solution in 2 ml of the Tris.HCI buffer was dialyzed for 3 days against 0.05 M KCI to remove traces of GTP. Poly(G) was obtained by treating the poly(G)-poly(dC) hybrid with deoxyribonuclease in 0.02 M Tris-HCI (pH 7.5) and 0.001 M MnCI2. After dialysis for 24 hr against the same buffer at room temperature, the Mn salt of poly(G) was collected by centrifugation and taken up in 0.001 M EDTA. Oligoadenylate. Radioactive oligo(A) with a free terminal 3' hydroxyl was prepared by partial hydrolysis of the hybrid of poly(dT) with poly(['4C]A) (7000 cpm/nmol), described before, with the aid of ribonuclease H. The incubation mixture (8 ml) consisted of 25 mM MgCl2, 0.05 mM KCI, 250 '4g/ml of bovine serum albumin, a quantity of the hybrid poly(dT)-poly(A) corresponding to 187.5 nmol/ml of adenylic acid, 125 units/ml of ribonuclease H (1), with the enzyme activity determined in the presence of Mg2+, in 0.05 M Tris-HCI of pH 8.0. The incubation mixture without the enzyme was brought to 350 and the reaction was then started by the addition of the enzyme. After 15 min at 350, the
1088
Biochemistry: Stavrianopoulos et al.
mixture was adjusted to pH 2.0-2.5 with 1 M HCl, thus inactivating the enzyme, and left at this pH for 10 min. Following neutralization with 1 M Tris base and adjustment to 37.5 mM EDTA, the solution was applied to a column of 2 ml of hydroxylapatite equilibrated with 0.15 M KCL. Elution was performed with potassium phosphate buffer of pH 6.8: 0.025 M for the oligonucleotides and 0.3 M for the unhydrolyzed hybrid. The 0.025 M eluate was placed on a column of 0.5 ml of DEAE-cellulose equilibrated with 0.05 M Tris.HCl of pH 8.0.t The small oligonucleotides (n < 5) were eluted with 8 ml of 0.2 M KC1 (flow rate 4 ml/hr) and the oligonucleotides of an average length of n = 7.2 were eluted with 0.4 M KCl at the same flow rate, the first 300 Mtl of eluate being discarded. Four fractions each of 0.5 ml were collected, of which the first three contained 4.561 X 106 cpm or 43.4% of the poly([14C]A) submitted to enzymic hydrolysis; the fourth eluate fraction corresponded to only 3% of the original radioactivity and was discarded. The proportion of radioactivity precipitable by trichloroacetic acid was determined 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, 14.3%. The average length of the oligo(A) preparation was estimated by alkaline hydrolysis (0.66 M LiOH, 370, 18 hr) and determination of adenosine, 2'(3')-adenylic acid, and adenosine 3',5'-diphosphate (paper chromatography in 1propanol/concentrated ammonia/water, 11:2:7, vol/vol/ vol). Since the length was found as 7.2 (based on pAp) or 7.5 (based on adenosine), this preparation is designated as (A)7.* Hybrid of Poly[(AHdA~J and Poly(dT). This copolymer hybrid was made by the action of the DNA polymerase of chicken embryo (2) obi the hybrid of poly(dT) with the 14Clabeled ribo oligomer p(A)7 in the presence of dATP: The incubation mixture (12 ml) consisted of 1.25 mM MnCl2, 83 mM KC1, 1.7 mM dithiothreitol, 400,uM dATP, 500,MM poly(dT), 50 MuM (A)7, 33 units/ml of DNA polymerase, and 250 ug/ml of bovine serum albumin, in 0.05 M Tris.HCl of pH 8.0. In order to facilitate a uniform distribution of the oligoadenylate on the poly(dT) present in a 10-fold excess, the following procedure was employed. A solution of the (Ah and the poly(dT) in 7 ml of 0.116 M KCl was kept at 700 for 10 min and then slowly brought to room temperature, the rest of the additives (minus enzyme) in 4.9 ml of buffer was admixed in an argon atmosphere, and the mixture was cooled to 00. Then the enzyme was added and incubation was performed at 200 for 12 hr (compare Table 1 in ref. 4). The radioactivity and its precipitability with trichloroacetic acid at zero time and at the end of the incubation were measured in duplicate in 50 gl portions. Per 50 Ml portion there were 17,181 cpm, of which 1226 (7.1%) were precipitable at zero time and 14,275 (83.1%) were precipitable at the end of the reaction. In order to remove unincorporated oligonucleotides, the incubation mixture was adjusted to 2.0 mM EDTA and to pH 7.0 by M Tris.HCI and then placed on a column of 1.5 ml of hydroxylapatite equilibrated with 0.15 M KCI. The unpolymerized oligonucleotides were eluted with 0.05 M potassium phosphate of pH 6.8, followed by the elution with t
Microcolumns are prepared conveniently by using the polypropylene tip of a 1 ml Eppendorf automatic pipette closed with a small plug of glass wool. The average chain length of these products is, of course, not entirely the same in different experiments. For instance, in an assay in which poly([3H]A)-poly(dT) was treated with ribonuclease H by a somewhat different procedure, the resulting oligo(A) had an average chain length of 8.9.
Proc. Nat. Acad. Sci. USA 73 (1976) 0.5 M potassium phosphate (pH 6.8) of the hybrid poly(dT)-
poly[(A)7-(dA),]. The balance of radioactivity (as nmol of [14C]adenylic acid) was: placed on a column, 489.4; eluted as oligonucleotides, 88.3 (18.0%); eluted as polymer hybrid, 314.9 (64.3%). A portion, 17.6% of the radioactivity, was not
eluted, presumably held back on the column as an enzymepolynucleotide complex. The 0.5 M potassium phosphate eluate of the polymer hybrid was freed of phosphate by being passed through a Sephadex G-25 column equilibrated with 0.1 M KCl or 0.2 M ammonium sulfate. This hybrid was completely resistant to pancreatic ribonuclease, as shown by the amount of radioactivity precipitable with 10% trichloroacetic acid at zero time and after enzyme treatment. In parallel experiments (in each: 55 gl solution, 0.1 M KCI, 0.01 M EDTA, pH 7.5, 15,000 cpm, 100 Mug of ribonuclease, 350, 30 min), the radioactivity precipitable at zero time was 14,930 and 15,050 cpm, and after ribonuclease treatment, 15,070 and 14,540 cpm. RESULTS Factors Influencing the Action of Ribonuclease H on
Homopolymer Hybrids. The base composition of the ribo strand, the length of the deoxyribo strand, the nature and the concentration of the metal activator, and also the salt concentration of the medium (left out of consideration here), all these determine, though to a varying degree, the course and the outcome of the enzymic action. Base Composition of Ribo Strand. Poly(dT)-poly(A), in the presence of Mg2+, is the best substrate, resembling the fi DNA-RNA hybrid (1:1) discussed previously (1) in its hydrolysis requirements. Poly(dC)-poly(G) is attacked much more slowly, requiring four times as long for the same degree of splitting as poly(dT)-poly(A) in the presence of Mn2+. The results summarized in Table 1 illustrate the variety of conditions governing the enzymic hydrolysis of the homopolymer hybrids under study. The fl hybrid mentioned before is included for comparison. The normalization of all results, required for comparison, is also explained in this table. Length of Deoxyribo Strand. As is also shown in Table 1, the degradation velocity rises with the increasing length of the deoxyribo strand. It is, however, remarkable that even deoxyribonucleotides as short as tetra offer sufficient support for the ribo strand to be attacked by the hybrid-specific enzyme, since the temperature of the enzyme reaction, 350, presumably lies above the melting temperatures of the respective double structures (6). It would not be surprising if the stability of the hydrogen-bonded alignment of the two strands were, in fact, increased by the enzyme as it proceeds to degrade the hybrid. The study of the effect of deoxyribo tetranucleotides, of which all four representatives could be examined (Exps. 1, 3, 6, and 9 in Table 1), offers an opportunity of comparing the influence of the particular base on the hydrolysis of the complementary ribo strand. The efficiency decreases in the following order: (dA)4.poly(U); (dG)4. poly(C); (dC)4.poly(G); (dT)4.poly(A), when Mn2+ is the activator. Pyrimidine ribo strands are broken more readily than purine strands. The only deoxyribo dinucleotide available, d(pApA), did not support the enzymic degradation of the complementary polyribo strand (Exp. 5). Divalent Metal Ions. The differential effects are somewhat baffling. Mn 2+ activates the attack on all hybrid combinations; Mg2+, only on those containing purine ribo strands; Co2+, only on poly(A) hybrids, but less well than
Biochemistry: Stavrianopoulos et al.
Proc. Nat. Acad. Sci. USA 73 (1976)
Table 1. Degradation efficiency of different hybrid substrates by ribonuclease H*
Components of equimolar hybrid Ribo Deoxyribo 1 2 3 4 5 6 7 8 9 10
(dT)4 Poly(dT)
(dC)4 Poly(dC) (dA)2
(dA)4
(dA), Poly(dA)
(dG)4 fl DNA
Poly(A) Poly(A) Poly(G) Poly(G) Poly(U) Poly(U) Poly(U) Poly(U) Poly(C) RNA tran-
script
Mg2+ 25 100 7.8 12.5 Inact. Inact. Inact. Inact. Inact.
66.5
% degradation Mn2+ C02+ 6.3 40 6.4 10.4
2.1 70 Inhib. Inhib. Inact. Inhib. Inhib.
Inact. 17.5 28.2
Inhib.
62.5
Inhib.
12.5
61.7
54.3
Each assay sample (120 ul) consisted of 0.05 M Tris-HCl (pH 8.0), of hybrid substrate corresponding to 83.3 MM with respect to the ribo moiety, of 0.4 mg/ml of bovine serum albumin, and of the additions specified below for each experiment. The indicated 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 mM MnCl2, 135 ng of enzyme, 0.1 M (NH4)2S04; 2.5 mM CoC12, 420 ng of enzyme, 0.2 M KCl.-Exp. 2: 25 mM MgCl2, 10 ng of enzyme, 0.05 M KQl; 1.5 mM MnCl2, 20 ng of enzyme, 0.2 M (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 enzyme.-Exp. 4: 10 mM MgCl2, 70 ng of enzyme, 0.05 M KQl; 0.25 mM MnCl2, 80 ng of enzyme, 0.15 M KCl.-Exps. 5 and 6: 10 mM MnCl2, 50 ng of enzyme.-Exp. 7: 10 mM MnCl2, 32 ng of enzyme, 0.1 M KCl.-Exp. 8: 1.2 mM MnCl2, 14 ng of enzyme, 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 splitting recorded in Exp. 2 in presence of Mg2+ was taken as 100%. In all other assays, performed under the optimum conditions listed above, the degradation values obtained were reduced by computation to those corresponding to 10 ng of enzyme per assay and the results were expressed as % of those obtained in Exp. 2 (Mg2+). "Inactive" means that this cation was inactive by itself and that its addition did not affect the activity of an active metal ion. "Inhibiting" means that the latter was depressed by the former.
*
Mg2+. With all other homopolymer hybrids, the cobalt ion fact, as an inhibitor. The metal ion concentrations showing optimum activity or 100% inhibition are summarized in Table 2. Manganese would seem to be the "normal" activator of the nuclease; it is the only one permitting the hydrolysis of ribo pyrimidine tracts. In order to exclude the possibility that the inhibition by Co2+ was merely an artifact of the analytical method employed, by aggregating degradation products, so as to render them no longer filtrable, a series of experiments was performed in which poly(dC)-poly(G), poly(dA)-poly(U), and (dG)4.poly(C) were treated with the enzyme in presence of Mn2+. At the end of the incubation, Co2+ was added to an 8 mM concentration, and then the mixtures were analyzed as usual. Exactly the same hydrolysis values were found as when Co2+ was omitted. The inhibition, therefore, is not illusionary. Enzymic Degradation of Poly[(A)dA~J.Poly(dT) Hybrid. The results of two experiments are described here, one with Mg2+, the other with Mn2+ as activator. The experimental conditions were as follows. Exp. 1: total volume 1.1 acts, in
Table 2. Effects of metal ions on ribonuclease H* Metal ion (mM)
Effect of metal on
1089
Components of equimolar hybrid Deoxyribo
Ribo
(dT)4 Poly(dT) (dC)4 Poly(dC) (dA)4
Poly(A) Poly(A) Poly(G)
(dA)s Poly(dA) (dG)4 fl DNA
Poly(G) Poly(U) Poly(U) Poly(U) Poly(C)
Optimum activity C0o2+ Mn2+ Mg2+ 1,5 1.5 0.4 0.4 10 10
10 25 12 10 Inact.
1.5 7.5
Inact. Inact. Inact.
1.5
25
100% Inhibition
Co2+
2.5 20 8 8 1.5 1.5
1.5 1
RNA tran-
script
20
*The experimental conditions were those indicated in Table 1, with the enzyme and salt concentrations specified there. The concentrations of metal activators or inhibitors varied, however, over the range shown here.
ml; 0.05 M Tris-HUl, pH 8.0; 0.025 M MgC12; 0.05 M KCl; 500 .g of bovine serum albumin; copolymer hybrid corresponding to 51.4 nmol of riboadenylic acid; and 500 units of ribonuclease H.-Exp. 2: total volume 1.3 ml; 0.05 M TrisHC1, pH 8.0; 2.0 mM MnCl2; 0.2 M (NH42S04; 500 ,ug of bovine serum albumin; copolymer hybrid corresponding to 53.3 nmol of riboadenylic acid; and 500 units of ribonuclease H. The solutions were incubated at 350 until the proportion of radioactivity precipitable by 10% trichloroacetic acid showed no more drop. This point was reached after an incubation of 1 hr when about '7 of the radioactivity submitted to enzymic digestion still could be precipitated. At this point % volume of 50% trichloroacetic acid was added to each vessel, the mixtures were kept in an ice bath for 30 min and centrifuged in the HB-4 rotor of the Sorvall centrifuge (2000 X g, 10 min). The sediments were washed by suspension in 0.5 ml of cold 10% trichloroacetic acid and by centrifugation, carefully drained and taken up, each in 100 gl of 0.66 M lithium hydroxide. After the addition of 200 ,g of 0.33 M LiOH to each sample, hydrolysis was performed at 370 for 18 hr. The procedures for analysis and chromatography were the same as those described before for the determination of the chain length of oligo(A). In Exp. 1, 14.5% of the radioactive riboadenylic acid
linked covalently to poly(dA) in the copolymer hybrid remained precipitable after treatment with ribonuclease H; in Exp. 2 the corresponding figure was 14.0%. The balance of the chromatographically separated products of the alkaline hydrolysis was (as % of total recovered material): in Exp. 1, adenosine 3',5'-diphosphate, 91; adenosine 2'(3')-monophosphate, 9. The corresponding values in Exp. 2 were 92% for pAp and 8% for Ap. This means that after the exhaustive treatment of hybrid poly[(A)7-(dA)1].poly(dT) with very large amounts of ribonuclease H the bulk of the resulting poly(dA) (82-84%) retained one riboadenylic acid end group, whereas a much smaller proportion (16-18%) still remained linked, on the average, to a ribo dinucleotide. Inhibiting Action of SAdenosylmethionine and SAdenosylhomocysteine. In our previous publication (1) we de-
1090
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Stavrianopoulos et al.
Table 4. Activity of ribonuclease H after preincubation with AdoMet or AdoHcy*
Table 3. Inhibition by S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy)* Substrate
Inhi-
0 95 81 0 97 80 0
-
7.14 0.34 1.37 6.14 0.2 1.2 5.6 0.25 4.9 5.6 0.19
22.5
5.07
Inhibitor (mM)
Conditions AdoMet 1 2 3 4 5 6 7 8 9 10 11 12 *
a a a
b b b
AdoHcy
8.0
-
-
22.5
8.0 22.5
c c
8.0
22.5
c
d d
d
8.0
96
12 0
97 9
Uniform experimental conditions: incubation for 15 min at 350; total volume, 0.12 ml; 0.05 M Tris.HCl, pH 8.0; 400 Mg/ml of bovine serum albumin; substrate, 83.3 MM with respect to the ribonucleotide moiety; indicated quantities of inhibitor. Varying 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: poly(dA).poly(U) as substrate; 1.5 mM MnCl2, 0.1 M KCI, 14 ng of enzyme; d: (dG)4.poly(C) as substrate; 10 mM MnCl2, 70 ng of enzyme. Compare footnote, §, for the inhibition tests with AdoHcy.
scribed briefly the observation that the activity of ribonuclease H is inhibited by S-adenosylmethionine (AdoMet). This effect could have been explained by the assumption that a contaminating protein O-methyltransferase (EC 2.1.1.24), which is known to occur in calf thymus (7), could have inactivated our enzyme by methylation. This explanation is, however, rendered less likely by the finding that S-adenosylhomocysteine (AdoHcy), although in 3fold concentration, also acts as an inhibitor of the nuclease, particularly with substrates containing purine ribopolymers.§ A selection of inhibition experiments is presented in Table 3. They show that 8 mM AdoMet completely blocks the enzymic degradation of all homopolymer hybrids, whereas 22.5 mM AdoHcy interferes effectively only with the breakdown of substrates containing poly(A) or poly(G). Other differences between these inhibitors are brought out in Table 4. By preincubation at 350 in the absence of substrate the enzyme is irreversibly inactivated by AdoMet, whereas AdoHcy has little effect. If the contact with the enzyme takes place at 00, however, neither AdoMet nor AdoHcy proves inhibitory. What is worthy of note is that the simultaneous pretreatment of the enzyme at 350 with both inhibitors appears to protect the enzyme partially against inactivation by AdoMet. DISCUSSION
The action of ribonuclease H is greatly affected by the composition of the substrate and the nature of the metal activa§
InhiInhibitor (mM) Substrate Temp. of hydrolyzed, bition, preincugmol % bation AdoMet AdoHcy
hydrolyzed, bition, Mrmol %
Because of the very limited solubility of S-adenosylhomocysteine (AdoHcy) in water, special precautions had to be followed in the preparation of assay mixtures containing this substance. A 30 mM solution of AdoHcy in water was prepared by placing the aqueous suspension in a completely dust-free tube and heating it to 85° until all was dissolved. The solution was then brought to pH 7.8 by the addition of 1 M Tris-HCI and stored at 350, under which conditions it remained clear for several hours. The final assay mixture (120 Ml) was made by mixing three portions: 90 A130 mM AdoHcy, 10 gl enzyme, 20 Ml containing all other components.
1 2 3 4 5 6 *
00 00
8.0
350 35°. 350
8.0
350
8.0
22.5 22.5
6.89 6.52 6.75 0.92 5.95 3.09
0 5 0 86 12 54
The substrate was poly(dT).poly(A). For the preparation of 30 mM AdoHcy, compare footnote, §. The experimental conditions of preincubation were: total volume, 120 Al; 0.05 M Tris.HCI, pH 8.0; 25 mM MgCl2; 0.05 M KCl; 400 MLg/ml of bovine serum albumin; 120 ng of ribonuclease H; indicated quantities of inhibitor; incubation for 5 min at 00 or 350. After the preincubation, 10 Mu portions of the mixtures (corresponding to 10 ng of enzyme) were added to 110 Ml of the assay mixture, containing the listed components (but without enzyme and inhibitor), supplemented with poly(dT).poly(A) (83.3 uM with respect to adenylic acid); incubation at 35° for 15 min.
tor. (The equally great influence of the ionic milieu is left out of consideration, since only the optimum conditions are specified in this paper.) Tables 1 and 2 illustrate the correla-
tion between activating metal ion and the composition of the ribo moiety of the hybrid substrate. Only the hydrolysis of hybridized poly(A) is catalyzed by cobalt, that of both purine polyribonucleotides is catalyzed by magnesium, whereas manganese is active with all substrates. These findings could conceivably lead to a differential method for the specific cleavage of a DNA-RNA hybrid. The fi DNA-RNA hybrid studied before (4) is cleaved in the presence of each of the three metal activators (Table 1; see also ref. 1), but possibly into fragments having different end groups according to the metal employed. The enzyme appears to require the presence of both a deoxyribo and a ribo strand. Poly(A)-poly(U) is not cleaved (8), but a systematic study of double-stranded polyribonucleotides under a variety of conditions may be interesting. It is likely that ribonuclease H must recognize, and attach itself to, a deoxyribo strand, in order to break the complementary polyribonucleotide. This points to the presence in the enzyme of at least two active centers. Asr short a deoxyribo component as a tetranucleotide suffices to form hybrids with a polyribo strand susceptible to enzymic cleavage (Table 1). That this occurs at lower salt concentrations than the attack on an all-poly hybrid may indicate that in the first case it is the conformation of the ribo strand that can impose itself on the array of short deoxy chains, producing conditions sufficient for enzymic hydrolysis. The existence, under the conditions of our experiments, of triple strands, of the form 2 oligo(dM)-poly(N), is most improbable, since these complexes require high salt concentrations (9, 10). The hybrid between poly(dA) and poly(U) is interesting: the equimolar hybrid is unstable, and this complex exists as poly(dA).2 poly(U) (10, 11). It is, in fact, this hybrid which is split by the enzyme most efficiently in the presence of Mn2+ (Exp. 8 in Table 1), possibly owing to the existence of a triple structure. The hybrid between poly(dT) and the block copolymer poly[(Ah-(dA)x] could be regarded as a model of the type of product formed in the replication process when a DNA polymerase is primed by a short RNA chain (12). Ribonuclease H acting on this hybrid formed degradation products of
Biochemistry: Stavrianopoulos et al.
Proc. Nat. Acad. Sci. USA 73 (1976)
which the bulk (83%) of the poly(deoxyadenylic -acid) moieties retained one riboadenylic acid end group, whereas 17% were linked to a ribodinucleotide.l In this respect the calf thymus enzyme resembles the corresponding enzyme from E. coil (13). This finding is of interest, since it predicts that under biological conditions a polymer of the type p(rM)m-(dN)n, which is bonded to a complementary all-deoxy strand, will be attacked by ribonuclease H in such a manner as to retain The remaining mono- or dithe grouping prM-dNp ribonucleotide residue would then have to be removed by other mechanisms unless it is retained in the completed DNA molecule. Preliminary experiments, not detailed in this paper, have made it likely that also in the counterpart of the hybrid discussed in the preceding paragraph, namely, in a hybrid between poly(dT) and p(dA)8-(A)2, one of the two ribonucleotides is removed by the enzyme, leaving, on the average, one riboadenylic acid residue linked 5' 3' to the octadeoxyrbonucleotide. Turning to the inhibition experiments described here, it will be noticed that cobalt ions, which activate the enzymic cleavage of poly(A) hybrids, inhibit that of all other homopolymer substrates (Table 2), most probably owing to the effect of the metal on the configuration of the substrates rather than of the enzyme. A more specific inhibitory effect, namely, of S-adenosylmethionine, was mentioned before (1). It is shown in more detail in Tables 3 and 4. This substance inactivates the enzyme irreversibly on preincubation at 350, but not at 00. The assumption that inactivation may be due to enzymic methylation would seem to be contradicted by the observation that S-adenosylhomocysteine in a higher concentration also acts as an inhibitor, although not irreversibly. Partial protection from irreversible inactivation is afforded by simultaneous treatment with both nucleoside derivatives, which may mean that both inhibitors compete for the same site on the enzyme. Since relatively high concentrations (6-8 mM) of S-adenosylmethionine are required for the inactivation of ribonuclease H-nearly the thousandfold amount of what is needed for enzymic methyl transfer (14)-even the nonenzymic methylation of the enzyme could be envisioned. ...
....
--
I
If an oligoribonucleotide is not stabilized by covalent 3' 5' linkage to a polydeoxyribonucleotide, as in this experiment, ribonuclease H produces fewer breaks. For instance, a hybrid between poly(dT) and (A)7 yielded, on treatment with large amounts of the enzyme, fragments of an average length of 3, presumably because even one break resulted in pieces that were too short to maintain the stable hybrid structure that is requisite for enzymic cleavage. -
1091
Ribonucleases specific for the cleavage of RNA in hybrid form are distributed very widely. It is probable that they have a significant function in replication or transcription. As concerns the first process, the enzyme may be operative in removing the RNA primer portion of a newly synthesized DNA chain (15). Once this is accomplished, the complementary DNA segment is left unpaired and available either for a repair process by DNA polymerase and ligase or as an initiator site for RNA polymerase B, which is known to prefer single-stranded DNA (16). If the transcription by this enzyme, bound initially to an unpaired DNA region produced as described here or in another manner, continues into the double-stranded portion of the DNA, the stretch of RNA transcribed from the latter will be extruded but remain attached, as an initial hybrid, to the initiator site. This hybrid segment again is a substrate for ribonuclease H, which thus would effect the release of the nonhybridized part of the RNA transcript. In this manner, the participation of ribonuclease H in the production, for instance, of heterogeneous nuclear RNA could be envisioned. This work was supported by U.S. Public Health Service Grant no. CA-12210. 1. Stavrianopoulos, J. G. & Chargaff, E. (1973) Proc. Nat. Acad. Sci. USA 70, 1959-1963. 2. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) Proc. Nat. Acad. Sci. USA 69,1781-1785. 3. Kato, K., Goncalves, J. M., Houts, G. E. & Bollum, F. J. (1967)
J. Biol. Chem. 242,2780-2789. 4. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) Proc. Nat. Acad. Sci. USA 69,2609-2613. 5. Farber, F. E. & Chargaff, E. (1967) Eur. J. Biochem. 2, 433441. 6. Uhlenbeck, O., Harrison, R. & Doty, P. (1968) in Molecular Associations in Biology, ed. Pullman, B. (Academic Press, New York and London), pp. 107-114. 7. Paik, W. K. & Kim, S. (1968) J. Biol. Chem. 243,2108-2114. 8. Hausen, P. & Stein, H. (1970) Eur. J. Biochem. 14,278-283. 9. Rich, A. (1960) Proc. Nat. Acad. Sci. USA 46,1044-1053. 10. Riley, M., Maling, B. & Chamberlin, M. J. (1966) J. Mol. Biol.
20,359-389. 11. Felsenfeld, G. & Rich, A. (1957) Biochim. Biophys. Acta 28, 457-468. 12. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1971) Proc. Nat. Acad. Sc. USA 68, 2207-2211. 13. Leis, J. P., Berkower, I. & Hurwitz, J. (1973) Proc. Nat. Acad. Sci. USA 70,466-470. 14. Jamaluddin, M., Kim, S. & Paik, W. K. (1975) Biochemistry 14,694-698. 15. Keller, W. (1972) Proc. Nat. Acad. Sci. USA 69,1560-1564. 16. Chambon, P. (1974) in The Enzymes, ed. Boyer, P. D., (Academic Press, New York and London), 3rd ed., Vol. X, pp. 261-331.
