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Stabilization of nucleic acids by proteins toward enzymic digestion
BIOCHIMICA ET BIOPHYSICA ACTA
51
BBA 95880
STABILIZATION OF NUCLEIC ACIDS BY P R O T E I N S TOWARD ENZYMIC DIGESTION J O S E P H D. PADAYATTY, MAX D. H E N S L E Y AND HAROLD VAN K L E Y
Department o[ Microbiology and Edward A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, St. Louis, Mo. (U.S.A.) (Received November 6th, 1967) (Revised manuscript received January 2nd, 1968)
SUMMARY
Ribonuclease and deoxyribonuclease were less active towards RNA and DNA, respectively, in the presence of proteins, especially fractionated histones. Basic proteins complex with nucleic acids and stabilize them against digestion by nucleases. Stabilization depends on the nature of the protein; the effect was lost when the protein was acetylated. Basic proteins reduce RNA synthesis in a rat liver nuclei RNA polymerase system, at the same time protect the newly synthesized RNA against ribonuclease action.
INTRODUCTION
Histones, the basic proteins present in the nucleus, are thought to be involved in the regulation of nucleic acid synthesis and cell division. 1 The mechanism of the role of histones in the nucleus is not clear, but they seem to inhibit many biosynthetic reactions including RNA synthesis, while removal of nuclear histones increases RNA synthesis. 2 POGO, ALLFREY AND MIRSKY3 have shown that acetylation of the basic protein of chromosomes appears to precede the increase in nuclear RNA synthesis. Besides the regulatory role of histones, they may be involved in stabilizing the nucleic acids toward degradation by nucleases which are present in abundant quantities in all cells. This is in agreement with the fact that there is little gross degradation of nucleic acids in most functioning cells except messenger RNA. To test this function of histones, the action of deoxyribonuclease on DNA and ribonuclease on RNA was studied in the presence of various basic proteins and acetylated basic proteins. Basic proteins stabilized DNA toward deoxyribonuclease and RNA toward ribonuclease, but the protecting ability was lost when the protein was acetylated. The inhibitory as well as the stabilizing action of histones and acetylated histones in the presence of ribonuclease were also investigated in a RNA polymerase system. It was found that all proteins tested, acetylated or not, show some protective action on the newly synthesized RNA toward ribonuclease. MATERIALS AND METHODS Histone (calf thymus-fractionated and unfractionated), DNA (calf thymus), RNA, deoxyribonuclease, pancreatic ribonuclease (Worthington Biochemical Corp., Biochim. Biophys. Acta, 161 (1968) 51-55
52
J. D. PADAYATTY, M. D. H E N S L E Y , H. VAN K L E Y
Freehold, N.J.); bovine serum albumin (Pentex rnc., Kankakee, Ilk); polylysine (New England Nuclear Corp., Boston, Mass.); protamine sulfate (arginine content 67-7o %, Nutritional Biochemicals Corp., Cleveland, Ohio); nucleoside triphosphates (Sigma Chemical Co., St. Louis, Mo.); [3HICTP and E3HIUTP (Schwarz BioResearch, Inc., Orangeburg, N.Y.) were used. Polyglutamyl serum albumin was a generous gift from Dr. 317. A. Stahman, University of Wisconsin. Proteins were acetylated according to the method of COLLIPP et al. 4 with some modifications. Protein (less than 4.5 rag) was dissolved in o. 5 ml water, and o.5 ml of saturated sodium acetate solution was added. The solution was cooled in an ice b a t h and excess acetic anhydride (o.o3 ml) was added in 3 portions over a period of 45 min. The sample was allowed to stand for 15 min and then dialyzed for 48 h with 3 changes of (demineralized) water. Since the molecular weight of protamine sulfate was low, it was not dialyzed. The nondiffusible material was made up to contain I mg of protein per ml of solution.
RESULTS AND DISCUSSION
Histones cause precipitation of nucleic acids, especially at low salt concentrations. In order to study the effect of histones on the action of nucleases on nucleic acids, they have to be used at levels at which there was no precipitation as observed by the turbidity at 450 m/~. The m a x i m u m amounts of histone or acetylated histone ~.hich did not cause precipitation of nucleic acids were determined and shown in Table I. Ribonuclease was assayed by the method of D I C K M A N , _A_ROSKARAND KNOPE5 and deoxyribonuclease by the procedure of KUNITZ~, at m a x i m u m levels of histone that did not cause any precipitation. To RNA (200/~g) and histone or acetylated histone in I ml of o.I M Tris-HC1, o.I M KC1 (pH 7.4), pre-incubated at 37 ° for IO TABLE
I
MAXIMUM AMOUNTS OF HISTONE THAT DID NOT CAUSE PRECIPITATION WITH NUCLEIC ACIDS R N A (200 fig) a n d h i s t o n e o r a c e t y l a t e d h i s t o n e (12.5, 25, 5 ° or i o o / z g ) i n I m l of o. I M T r i s - H C 1 , o . I M KC1 ( p H 7.4), w a s i n c u b a t e d a t 37 ° for IO r a i n a n d t h e t u r b i d i t y a t 450 m / , w a s r e a d o n Zeiss PMQII Spectrophotometer. Similar experiments were carried out with DNA and histone a n d a c e t y l a t e d h i s t o n e i n 0.08 M s o d i u m a c e t a t e , o.o15 M M g 2+ ( p H 6.5). F r o m a p l o t of t h e w e i g h t s of h i s t o n e o r a c e t y l a t e d h i s t o n e a g a i n s t t u r b i d i t y a t 450 m/z a n d e x t r a p o l a t i o n to z e r o t u r b i d i t y , t h e m a x i m u m a m o u n t s of h i s t o n e o r a c e t y l a t e d h i s t o n e t h a t d i d n o t c a u s e p r e c i p i t a t i o n of n u c l e i c a c i d s w e r e d e t e r m i n e d .
Unfractionated histone Slightly lysine-rich histone Lysine-rich histone Arginine-rich histone Polylysine Protamine sulfate Bovine serum albumin Polyglutamyl bovine serum albumin
Protein (tzg)
A~etylated protein (Izg)
RNA
DNA
RNA
DNA
25 25 13 io 9 5 4° 4°
8 15 7 io 8 4
15 5 18 5 12 18
io 6 25 5 2 2
Biochim. Biophys. Acta, 161 (1968) 5 1 - 5 5
STABILIZATION OF NUCLEIC ACIDS BY PROTEINS
53
rain, ribonuclease (o.I ~g) was added and digested for I h at 37 °. 3.o ml tert.-butylalcohol-acetic acid (3: I, v/v) were added to the reaction mixture; the samples cooled in ice bath, centrifuged, and the absorbance of the supernatant solution at 258 m# was read. To DNA (2oozg) and protein or acetylated protein in I ml of 0.08 M sodium acetate o.o15 IV[ Mg i+ (pH 6.5), pre-incubated at 37 ° for IO min, deoxyribonuclease (0. 4 #g) was added. The samples were incubated at 37 ° for IO rain, then 3 ml of o.125 M H2SO 4 was added. Solutions were cooled in an ice bath, centrifuged, and the absorbance of the supernatant solution at 260 m# was read. There was a decrease in the activities of ribonuclease and deoxyribonuclease in the presence of histones. This could be a reflection of the inhibition of nucleases b y histones. However, this is not likely since nuclease activity was observed in the presence of a large excess of histone. The slight decrease in nuclease activity in the presence of histones m a y be due to the combination of histones and nucleic acids to form complexes which are not accessible to nucleases. This protective action against ribonuclease decreases from acetylated protamine sulfate (12.5 %), slightly lysinerich histone ( I I °/o), lysine-rich histone (8 °/o), arginine-rich histone (4 %), polylysine (2 %) to unfractionated histone (2 %). Bovine serum albumin and polyglutamyl bovine serum albumin had no effect. The protective effect of all proteins except protamine sulfate was removed b y acetylation. The effect of acetylated protamine sulfate was not clear because of the presence of excess salt in the preparation. It m a y be concluded that basic proteins stabilize the R N A and make it less sensitive to ribonuclease. This is in full agreement with the report 7 that the R N A of R N A histone complex was ribonuclease-insensitive when bound to histone but sensitive to the enzyme when removed from the protein. The stabilizing effect of histone towards the digestion of DNA b y deoxyribonuclease decreases from polylysine (15 °/o), arginine-rich histone (15 %), lysine-rich histone (9 %), slightly lysine-rich histone (3 %) to unfractionated histone (2 %). Protamine sulfate and acetylated proteins have no protective effect. Again, the basic proteins confer stability to DNA toward TABLE II INCORPORATION OF [3H]CTP INTO R N A IN PRESENCE AND ABSENCE OF RIBONUCLEASE AND PROTEINS OR ACETYLATED PROTEINS I n a d d i t i o n to t h e i n g r e d i e n t s d e s c r i b e d b y GORSKI s, t h e i n c u b a t i o n m i x t u r e c o n t a i n e d o.2 ml of t h e n u c l e a r p r e p a r a t i o n , IOO p g of p r o t e i n or a c e t y l a t e d p r o t e i n a n d o . 5 / l g of r i b o n u c l e a s e i n t h e s a m p l e s i n d i c a t e d . T h e i n c u b a t i o n p e r i o d w a s 3 ° m i n a t 37 °. The a c i d i n s o l u b l e d e f a t t e d r e s i d u e w a s d i s s o l v e d in I m l of I.O M h y d r o x i d e of h y a m i n e in m e t h a n o l ( w a r m e d a t 6o ° for 15-2o min), c o u n t e d in l i q u i d s c i n t i l l a t i o n c o u n t e r for 3-5 m i n a n d d i s i n t . / m i n w e re d e t e r m i n e d u s i n g i n t e r n a l standards.
Disint./min × zo -3 Protein
None Unfractionated histone Slightly lysine-rich histone Lysine-rich histone Arginine-rich histone Polylysine Protamine sulfate
Acetylated protein
No ribonuclease
Ribonuclease
No ribonuclease
Ribonuclease
13. 3 14.o 3.3 7.6 9.3 5-5 io.o
4.2 6.0 2.6 5.6 7-5 3 .8 7.9
13.3 12. 4 6. 4 8.7 8-5 9 .1 7.3
4.2 4.4 4.o 7.1 4.9 5.3 5 .o
Biochim. Biophys. Acta, 161 (1968) 51-55
54
J . D . PADAYATTY, M. D. HENSLEY, H. VAN KLEY
the action of deoxyribonuclease and the protective action was lost when the protein was acetylated. The stabilization of RNA by basic protein was studied by following RNA synthesis by the procedure of G-ORSKI8 with the addition of ribonuclease and protein or acetylated protein. Nuclei isolated from the liver of I-month-old male rat (St. Louis University rat colony) according to the procedure of WlDSELL AND TaTA 9 served as a source of RNA polymerase. The nuclei were suspended in 5 ml of o.25 M sucrose solution containing i.o m ~ magnesium chloride. There was a linear relationship between the incorporation of E3HJCTP or [~HIUTP into RNA and the quantity of nuclear preparation up to o. 4 ml (A4s0 ma = 1.o). The RNA synthesis was followed b y determining the incorporation of [3H]CTP into the acid-insoluble precipitate as shown in Table II. The incorporation of EaH]UTP into RNA was studied at different temperatures, using the nuclear material as a source of RNA polymerase prepared according to the method of BLOBEL AND POTTER1°. The results are shown in Fig. I.
400
-•300 vZ
u 200
10010
2JO
3'0
4'0
TEMPERATURE
Fig. I. Effect of t e m p e r a t u r e on the i n c o r p o r a t i o n of E~H]UTP into R N A b y a r a t liver nuclear preparation. I n c o r p o r a t i o n of [3H]UTP into R N A at different t e m p e r a t u r e s were determined according to the m e t h o d of GORSKI8, using 0. 3 ml of nuclear p r e p a r a t i o n as source of R N A polymerase, the i n c u b a t i o n period was 3° min. Trichloroacetic acid (io °/o, 2 ml) was added, cooled in ice b a t h and filtered t h r o u g h a millipore filter, t h e n w a s h e d t h o r o u g h l y with 5 °/o cold trichloroacetic acid. The r a d i o a c t i v i t y on the millipore filter was counted in liquid scintillation counter.
The incorporation of the label was maximum at range 2o ° to 28 °, the incorporation at 37 ° was only 57 % of that at 25 °. In order for our data to be comparable to others in the literature, the experiments were run at 37 °. The incorporation of [aH]UTP into RNA in the presence and absence of ribonuclease and histones or acetylated histones were determined as under Table II, the trichloroacetic acid-insoluble precipitate was washed over millipore filters and the radioactivity on the filters was counted. The effect of protein and acetylated protein on the action of ribonuclease towards newly synthesized RNA were similar to that of the results obtained in Table II. RNA synthesis was inhibited 25-80 % in the presence of protein except unfractionated histone, the degree of inhibition was dependent on the nature of the protein. BARR AND BUTLERn have also found that DNA-histone complexes were active in the R N A polymerase system while the various fractions of histones were inhibitory. When acetylated protein was used, the inhibition was 32-53 %, Biochim. Biophys. Acta, 161 (1968) 51-55
STABILIZATION OF NUCLEIC ACIDS BY PROTEINS
55
showing acetylation reduced the inhibitory action. In the absence of any protein, 69 % of the newly synthesized RNA was digested by ribonuclease. In the presence of protein 19-31% and in the presence of acetylated protein 12-42 % of the newly synthesized RNA was digested by ribonuclease. Unfractionated histone or its acetylated form show little protective action since about 57 % of the newly synthesized RNA was digested by ribonuclease. This suggests that various fractions of histones, acetylated or not, stabilize newly synthesized RNA against digestion by ribonuclease. Alternatively, fractions of histones may inhibit proteolytic enzymes which would attack RNA polymerase and thereby increase RNA synthesis. The metabolic role of histones is complicated by the fact that basic proteins make the RNA insensitive toward ribonuclease and at the same time inhibit RNA synthesis by complexing with DNA.
ACKNOWLEDGEMENT
This work was supported by Cancer Research Institutional Committee Award Number 12 from funds supplied by the American Cancer Society and by U.S. Public Health Service. Grant Number GMIo6o4-o3 from the Institute of General Medical Sciences.
REFERENCES I J, BONNER AND 1~. C. HUANG, J. Mol. Biol., 6 (1963) 169. 2 V. G. ALLFREY, Cancer Res., 26 (1966) 2026. 3 B. G. T. POGO, V. G. ALLFRE¥ AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 55 (1966) 805. 4 P. J- COLLIPP, S. A. KAPLAN, D. C. BOYLE AND C. S. ~q'. SHIMIZU, J. Biol. Chem., 240 (1965) 143. 5 S. ]~. DICKMAN, J. P. AROSKAR AND R. B. KNOPF, Biochim. Biophys. Acta, 21 (1956) 539. 6 M. J. KUNITZ, J. Gen. Physiol., 33 (195 °) 363 • 7 W. BENJAMIN, O. A. LEVANDER, A. GELLHORN AND ]~. H. DEBELLIS, Proc. Natl. Acad. Sci. U.S., 55 (1966) 858. 8 J. GORSKI, J. Biol. Chem., 239 (1964) 889. 9 C. C. WIDNELL AND J. R. TATA, Biochem. J., 92 (1964) 313 • IO G. BLOBEL AND V. R. POTTER, Science, 154 (1966) 1662. i i G. C. BARR AND J. A. V. BUTLER, Nature, 199 (1963) 117o.
Biochim. Biophys. Acta, 161 (1968) 51-55
51
BBA 95880
STABILIZATION OF NUCLEIC ACIDS BY P R O T E I N S TOWARD ENZYMIC DIGESTION J O S E P H D. PADAYATTY, MAX D. H E N S L E Y AND HAROLD VAN K L E Y
Department o[ Microbiology and Edward A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, St. Louis, Mo. (U.S.A.) (Received November 6th, 1967) (Revised manuscript received January 2nd, 1968)
SUMMARY
Ribonuclease and deoxyribonuclease were less active towards RNA and DNA, respectively, in the presence of proteins, especially fractionated histones. Basic proteins complex with nucleic acids and stabilize them against digestion by nucleases. Stabilization depends on the nature of the protein; the effect was lost when the protein was acetylated. Basic proteins reduce RNA synthesis in a rat liver nuclei RNA polymerase system, at the same time protect the newly synthesized RNA against ribonuclease action.
INTRODUCTION
Histones, the basic proteins present in the nucleus, are thought to be involved in the regulation of nucleic acid synthesis and cell division. 1 The mechanism of the role of histones in the nucleus is not clear, but they seem to inhibit many biosynthetic reactions including RNA synthesis, while removal of nuclear histones increases RNA synthesis. 2 POGO, ALLFREY AND MIRSKY3 have shown that acetylation of the basic protein of chromosomes appears to precede the increase in nuclear RNA synthesis. Besides the regulatory role of histones, they may be involved in stabilizing the nucleic acids toward degradation by nucleases which are present in abundant quantities in all cells. This is in agreement with the fact that there is little gross degradation of nucleic acids in most functioning cells except messenger RNA. To test this function of histones, the action of deoxyribonuclease on DNA and ribonuclease on RNA was studied in the presence of various basic proteins and acetylated basic proteins. Basic proteins stabilized DNA toward deoxyribonuclease and RNA toward ribonuclease, but the protecting ability was lost when the protein was acetylated. The inhibitory as well as the stabilizing action of histones and acetylated histones in the presence of ribonuclease were also investigated in a RNA polymerase system. It was found that all proteins tested, acetylated or not, show some protective action on the newly synthesized RNA toward ribonuclease. MATERIALS AND METHODS Histone (calf thymus-fractionated and unfractionated), DNA (calf thymus), RNA, deoxyribonuclease, pancreatic ribonuclease (Worthington Biochemical Corp., Biochim. Biophys. Acta, 161 (1968) 51-55
52
J. D. PADAYATTY, M. D. H E N S L E Y , H. VAN K L E Y
Freehold, N.J.); bovine serum albumin (Pentex rnc., Kankakee, Ilk); polylysine (New England Nuclear Corp., Boston, Mass.); protamine sulfate (arginine content 67-7o %, Nutritional Biochemicals Corp., Cleveland, Ohio); nucleoside triphosphates (Sigma Chemical Co., St. Louis, Mo.); [3HICTP and E3HIUTP (Schwarz BioResearch, Inc., Orangeburg, N.Y.) were used. Polyglutamyl serum albumin was a generous gift from Dr. 317. A. Stahman, University of Wisconsin. Proteins were acetylated according to the method of COLLIPP et al. 4 with some modifications. Protein (less than 4.5 rag) was dissolved in o. 5 ml water, and o.5 ml of saturated sodium acetate solution was added. The solution was cooled in an ice b a t h and excess acetic anhydride (o.o3 ml) was added in 3 portions over a period of 45 min. The sample was allowed to stand for 15 min and then dialyzed for 48 h with 3 changes of (demineralized) water. Since the molecular weight of protamine sulfate was low, it was not dialyzed. The nondiffusible material was made up to contain I mg of protein per ml of solution.
RESULTS AND DISCUSSION
Histones cause precipitation of nucleic acids, especially at low salt concentrations. In order to study the effect of histones on the action of nucleases on nucleic acids, they have to be used at levels at which there was no precipitation as observed by the turbidity at 450 m/~. The m a x i m u m amounts of histone or acetylated histone ~.hich did not cause precipitation of nucleic acids were determined and shown in Table I. Ribonuclease was assayed by the method of D I C K M A N , _A_ROSKARAND KNOPE5 and deoxyribonuclease by the procedure of KUNITZ~, at m a x i m u m levels of histone that did not cause any precipitation. To RNA (200/~g) and histone or acetylated histone in I ml of o.I M Tris-HC1, o.I M KC1 (pH 7.4), pre-incubated at 37 ° for IO TABLE
I
MAXIMUM AMOUNTS OF HISTONE THAT DID NOT CAUSE PRECIPITATION WITH NUCLEIC ACIDS R N A (200 fig) a n d h i s t o n e o r a c e t y l a t e d h i s t o n e (12.5, 25, 5 ° or i o o / z g ) i n I m l of o. I M T r i s - H C 1 , o . I M KC1 ( p H 7.4), w a s i n c u b a t e d a t 37 ° for IO r a i n a n d t h e t u r b i d i t y a t 450 m / , w a s r e a d o n Zeiss PMQII Spectrophotometer. Similar experiments were carried out with DNA and histone a n d a c e t y l a t e d h i s t o n e i n 0.08 M s o d i u m a c e t a t e , o.o15 M M g 2+ ( p H 6.5). F r o m a p l o t of t h e w e i g h t s of h i s t o n e o r a c e t y l a t e d h i s t o n e a g a i n s t t u r b i d i t y a t 450 m/z a n d e x t r a p o l a t i o n to z e r o t u r b i d i t y , t h e m a x i m u m a m o u n t s of h i s t o n e o r a c e t y l a t e d h i s t o n e t h a t d i d n o t c a u s e p r e c i p i t a t i o n of n u c l e i c a c i d s w e r e d e t e r m i n e d .
Unfractionated histone Slightly lysine-rich histone Lysine-rich histone Arginine-rich histone Polylysine Protamine sulfate Bovine serum albumin Polyglutamyl bovine serum albumin
Protein (tzg)
A~etylated protein (Izg)
RNA
DNA
RNA
DNA
25 25 13 io 9 5 4° 4°
8 15 7 io 8 4
15 5 18 5 12 18
io 6 25 5 2 2
Biochim. Biophys. Acta, 161 (1968) 5 1 - 5 5
STABILIZATION OF NUCLEIC ACIDS BY PROTEINS
53
rain, ribonuclease (o.I ~g) was added and digested for I h at 37 °. 3.o ml tert.-butylalcohol-acetic acid (3: I, v/v) were added to the reaction mixture; the samples cooled in ice bath, centrifuged, and the absorbance of the supernatant solution at 258 m# was read. To DNA (2oozg) and protein or acetylated protein in I ml of 0.08 M sodium acetate o.o15 IV[ Mg i+ (pH 6.5), pre-incubated at 37 ° for IO min, deoxyribonuclease (0. 4 #g) was added. The samples were incubated at 37 ° for IO rain, then 3 ml of o.125 M H2SO 4 was added. Solutions were cooled in an ice bath, centrifuged, and the absorbance of the supernatant solution at 260 m# was read. There was a decrease in the activities of ribonuclease and deoxyribonuclease in the presence of histones. This could be a reflection of the inhibition of nucleases b y histones. However, this is not likely since nuclease activity was observed in the presence of a large excess of histone. The slight decrease in nuclease activity in the presence of histones m a y be due to the combination of histones and nucleic acids to form complexes which are not accessible to nucleases. This protective action against ribonuclease decreases from acetylated protamine sulfate (12.5 %), slightly lysinerich histone ( I I °/o), lysine-rich histone (8 °/o), arginine-rich histone (4 %), polylysine (2 %) to unfractionated histone (2 %). Bovine serum albumin and polyglutamyl bovine serum albumin had no effect. The protective effect of all proteins except protamine sulfate was removed b y acetylation. The effect of acetylated protamine sulfate was not clear because of the presence of excess salt in the preparation. It m a y be concluded that basic proteins stabilize the R N A and make it less sensitive to ribonuclease. This is in full agreement with the report 7 that the R N A of R N A histone complex was ribonuclease-insensitive when bound to histone but sensitive to the enzyme when removed from the protein. The stabilizing effect of histone towards the digestion of DNA b y deoxyribonuclease decreases from polylysine (15 °/o), arginine-rich histone (15 %), lysine-rich histone (9 %), slightly lysine-rich histone (3 %) to unfractionated histone (2 %). Protamine sulfate and acetylated proteins have no protective effect. Again, the basic proteins confer stability to DNA toward TABLE II INCORPORATION OF [3H]CTP INTO R N A IN PRESENCE AND ABSENCE OF RIBONUCLEASE AND PROTEINS OR ACETYLATED PROTEINS I n a d d i t i o n to t h e i n g r e d i e n t s d e s c r i b e d b y GORSKI s, t h e i n c u b a t i o n m i x t u r e c o n t a i n e d o.2 ml of t h e n u c l e a r p r e p a r a t i o n , IOO p g of p r o t e i n or a c e t y l a t e d p r o t e i n a n d o . 5 / l g of r i b o n u c l e a s e i n t h e s a m p l e s i n d i c a t e d . T h e i n c u b a t i o n p e r i o d w a s 3 ° m i n a t 37 °. The a c i d i n s o l u b l e d e f a t t e d r e s i d u e w a s d i s s o l v e d in I m l of I.O M h y d r o x i d e of h y a m i n e in m e t h a n o l ( w a r m e d a t 6o ° for 15-2o min), c o u n t e d in l i q u i d s c i n t i l l a t i o n c o u n t e r for 3-5 m i n a n d d i s i n t . / m i n w e re d e t e r m i n e d u s i n g i n t e r n a l standards.
Disint./min × zo -3 Protein
None Unfractionated histone Slightly lysine-rich histone Lysine-rich histone Arginine-rich histone Polylysine Protamine sulfate
Acetylated protein
No ribonuclease
Ribonuclease
No ribonuclease
Ribonuclease
13. 3 14.o 3.3 7.6 9.3 5-5 io.o
4.2 6.0 2.6 5.6 7-5 3 .8 7.9
13.3 12. 4 6. 4 8.7 8-5 9 .1 7.3
4.2 4.4 4.o 7.1 4.9 5.3 5 .o
Biochim. Biophys. Acta, 161 (1968) 51-55
54
J . D . PADAYATTY, M. D. HENSLEY, H. VAN KLEY
the action of deoxyribonuclease and the protective action was lost when the protein was acetylated. The stabilization of RNA by basic protein was studied by following RNA synthesis by the procedure of G-ORSKI8 with the addition of ribonuclease and protein or acetylated protein. Nuclei isolated from the liver of I-month-old male rat (St. Louis University rat colony) according to the procedure of WlDSELL AND TaTA 9 served as a source of RNA polymerase. The nuclei were suspended in 5 ml of o.25 M sucrose solution containing i.o m ~ magnesium chloride. There was a linear relationship between the incorporation of E3HJCTP or [~HIUTP into RNA and the quantity of nuclear preparation up to o. 4 ml (A4s0 ma = 1.o). The RNA synthesis was followed b y determining the incorporation of [3H]CTP into the acid-insoluble precipitate as shown in Table II. The incorporation of EaH]UTP into RNA was studied at different temperatures, using the nuclear material as a source of RNA polymerase prepared according to the method of BLOBEL AND POTTER1°. The results are shown in Fig. I.
400
-•300 vZ
u 200
10010
2JO
3'0
4'0
TEMPERATURE
Fig. I. Effect of t e m p e r a t u r e on the i n c o r p o r a t i o n of E~H]UTP into R N A b y a r a t liver nuclear preparation. I n c o r p o r a t i o n of [3H]UTP into R N A at different t e m p e r a t u r e s were determined according to the m e t h o d of GORSKI8, using 0. 3 ml of nuclear p r e p a r a t i o n as source of R N A polymerase, the i n c u b a t i o n period was 3° min. Trichloroacetic acid (io °/o, 2 ml) was added, cooled in ice b a t h and filtered t h r o u g h a millipore filter, t h e n w a s h e d t h o r o u g h l y with 5 °/o cold trichloroacetic acid. The r a d i o a c t i v i t y on the millipore filter was counted in liquid scintillation counter.
The incorporation of the label was maximum at range 2o ° to 28 °, the incorporation at 37 ° was only 57 % of that at 25 °. In order for our data to be comparable to others in the literature, the experiments were run at 37 °. The incorporation of [aH]UTP into RNA in the presence and absence of ribonuclease and histones or acetylated histones were determined as under Table II, the trichloroacetic acid-insoluble precipitate was washed over millipore filters and the radioactivity on the filters was counted. The effect of protein and acetylated protein on the action of ribonuclease towards newly synthesized RNA were similar to that of the results obtained in Table II. RNA synthesis was inhibited 25-80 % in the presence of protein except unfractionated histone, the degree of inhibition was dependent on the nature of the protein. BARR AND BUTLERn have also found that DNA-histone complexes were active in the R N A polymerase system while the various fractions of histones were inhibitory. When acetylated protein was used, the inhibition was 32-53 %, Biochim. Biophys. Acta, 161 (1968) 51-55
STABILIZATION OF NUCLEIC ACIDS BY PROTEINS
55
showing acetylation reduced the inhibitory action. In the absence of any protein, 69 % of the newly synthesized RNA was digested by ribonuclease. In the presence of protein 19-31% and in the presence of acetylated protein 12-42 % of the newly synthesized RNA was digested by ribonuclease. Unfractionated histone or its acetylated form show little protective action since about 57 % of the newly synthesized RNA was digested by ribonuclease. This suggests that various fractions of histones, acetylated or not, stabilize newly synthesized RNA against digestion by ribonuclease. Alternatively, fractions of histones may inhibit proteolytic enzymes which would attack RNA polymerase and thereby increase RNA synthesis. The metabolic role of histones is complicated by the fact that basic proteins make the RNA insensitive toward ribonuclease and at the same time inhibit RNA synthesis by complexing with DNA.
ACKNOWLEDGEMENT
This work was supported by Cancer Research Institutional Committee Award Number 12 from funds supplied by the American Cancer Society and by U.S. Public Health Service. Grant Number GMIo6o4-o3 from the Institute of General Medical Sciences.
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