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Change of nuclear ribonuclease H activity following deoxyribonucleic acid synthesis in liver of protein-deficient rat
Life Sclencee, Vol. 26, pp. 1497-1503 P¢InCed in the U.S.A.
Persamon Presl
CHANGE OF NUCLEAR RIBONUCLEASE H ACTIVITY FOLLOWING DEOXYRIBONUCLEIC ACID SYNTHESIS IN LIVER OF PROTEINDEFICIENT RAT Yaeuko Sawai, KenJi Wada and KinJi Tsukada Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo i01, Japan (Recelved In final form March 3, 1980) SUMMARY The activities of RNA polymerase I and Mg 2+dependent ribonuclease H (RNase H) become doubled in the liver nuclei of rats that are fed for several days a diet that lacks amino acids or protein. Both Mn 2+and Mg2+-dependent RNases H activities increased twoto three-~old in the liver nuclei of rats in which hepatic DNA replication has been induced by a dletal manipulation. Ribonucleaee H (RNaee H), which cleaves the RNA moiety of RNA-DNA hybrid molecules, is a ubiquitous enzyme in eukaryotlc, although the function of this enzyme still remains to be elucidated. A preceding report from our laboratory has shown that rat ~ liver contains at least two different RNames H a~ivities, an Me'-dependent RNase H of 150,000 daltons, and an Mg'~-dependent RName H of 35,000 daltons, distinguished from one another by physical and catalytic properties (i). The activity of nucleolar RNA polymorale increases in the hepatocytes of rats deprived of amino acids (2-4). In protein-deflcients, nuclear DNA synthesis and mitosis rasumm in some of the liver parenchymal cells soon after the animal is fed a meal containing protein (5) or some of the essential amino acids (6). The nucleolar changes including hypertrophy (79), an accumulation of RNA (10), and an increased activity of nucleolar RNA polymerase (2-4), characterize the liver of the amino acid-deficient rat as well as after partial hepatectomy. We have asked whether the activities of two nuclear RNasem H are elevated in the livers of rats induced to make DNA as well as RNA. This report describes that Mn2÷-dependent RNase H is involved in DNA synthesis, and Mg2÷-dependent RNase H is involved in synthesis of RNA and probably of DNA in liver nuclei. MATERIALS AND METHODS Male ~istar rats (140-150 g) were obtained locally. Unless otherwise specified, they were freely given protein-free diet and 50% casein to the time of the experiment. The protein-free diet was prepared by Teklad Mills, Madison Wisconsin, and consisted of 64% corn starch, 8% glucose, 10% corn oil, 4% "Salt Mix, U.S.A. XIV", 2% "Vitamin Fortification Mix" and 12% nonnutritive fiber. 002~-3205/80/181497-07$02.00/0 Copyrishc (c) 1980 PerSamon Prasm Lid
1498
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
Nuclei were i s o l a t e d from 10% h o m o g e n a t e of liver in s o l u t i o n c o n t a i n i n g only sucrose and CaCI 2 (ii). N u c l e i from 3 g of w e t liver were s u s p e n d e d in 1.5 ml of 0.15 M NaCl c o n t a i n i n g 5 m M 2m e r c a p t o e t h a n o l for 20 min at 2°C. The s u s p e n s i o n was c e n t r i f u g e d for 5 m i n at 10,000 x ~, the s u p e r n a t a n t fluid was a s s a y e d for RNase H a c t i v i t y and the p r e c i p i t a t e was used for assay of RNA p o l y m e r a s e I. RNA p o l y m e r a s e I was m e a s u r e d by its i n s e n s i b i l i t y of h i g h levels of ~ - a m a n i t i n w i t h liver nuclei by the m e t h o d s d e s c r i b e d p r e v i o u s l y (4). R a d i o a c t i v e l y l a b e l e d D N A - R N A h y b r i d was s y n t h e s i z e d on d e n a t u r e d calf thymus DNA using E.coli RNA p o l y m e r a s e as d e s c r i b e d p r e v i o u s l y (12). The assay of RNase H was based on the r e l e a s e into acid soluble fraction, of r a d i o a c t i v e m a t e r i a l from [3H]-RNA h y b r i d i z e d to DNA as d e s c r i b e d p r e v i o u s l y (13). P r o t e i n was d e t e r m i n e d DNA by the m e t h o d of B u r t o n
by the m e t h o d (15).
of Lowry
et al
(14)
and
RESULTS The s p e c i f i c a c t i v i t i e s of RNase H and RNA p o l y m e r a s e I in the n u c l e a r p r e p a r a t i o n s w e r e m e a s u r e d as a f u n c t i o n of time of d e p r i v a t i o n of amino acids. As shown in Table I, M g 2 + - d e p e n d e n t RNase H and RNA p o l y m e r a s e I rose g r a d u a l l y for several days. As c a l c u l a t e d from the Table, similar increase~ w e r e found in the a c t i v i t y of RNA p o l y m e r a s e I and that of M g 2 + - d e p e n d e n t RNase H, for the a c t i v i t y ratio of RNA p o l y m e r a s e I to M g 2 + - d e p e n d e n t RNase H r e m a i n e d c o n s t a n t for s e v e r a l days tested. Under these conditions, w h e n the e n z y m e a s s a y was p e r f o r m e d u s i n g Mn 2+, no significant d i f f e r e n c e in the level of RNase H a c t i v i t y was detected. A single m e a l c o n t a i n i n g p r o t e i n c a u s e d n u c l e a r DNA r e p l i c a t i o n in some of the h e p a t o c y t e s of the p r o t e i n - d e f i c i e n t rat (5, 6). As shown in Table I, s u p p l e m e n t a t i o n of the p r o t e i n - f r e e m a s h w i t h 50% casein and t h y r o i d h o r m o n e e f f e c t on the s u b s e q u e n t f o r m a t i o n of DNA, after 20 h. Under these conditions, b o t h Mg ~-- and Mn ~ d e p e n d e n t RNases H a c t i v i t i e s h a d i n c r e a s e d two- to t h r e e - f o l d w i t h i n 20 h (Table I). DNA f o r m a t i o n could be i n d u c e d in the liver of the p r o t e i n - d e f i c i e n t rat by feeding 15% c a s e i n Qr t h y r o i d h o r m o n e b u t the e f f e c t was smaller. The a c t i v i t i e s of Mg 2+- and M n 2 + - d e p e n d e n t RNases H w e r e r e d u c e d by such a treatment. Fig. 1 shows the p h o s p h o c e l l u l o s e c o l u m n c h r o m a t o g r a p h y Mn2+ p r o f i l e of n u c l e a r RNase H. Two m a j o r peaks c o r r e s p o n d i n g to and M g 2 + - d e p e n d e n t RNases H w e r e e l u t e d at a b o u t 0.2 M and 0.4 M KCI and a m i n o r p e a k h a v i n g RNase H a c t i v i t y was r e c o v e r e d in the f l o w - t h r o u g h f r o m2+ the column. As shown in" Fig. IB, the total activity of the Mg - d e p e n d e n t RNase H was about 2-fold h i g h e r at 5 days w i t h d e f i c i e n t rats, w h e r e a s the a c t i v i t y of M n 2 + - d e p e n d e D t RNase H w a s r e l a t i v e l y unaffected. However, both Mg 2+- and Mn 2+d e p e n d e n t RNases H a c t i v i t i e s w e r e i n c r e a s e d about 2- to 3-fold w h e n DNA s y n t h e s i s was induced by a meal of 50% c a s e i n and t h y r o i d h o r m o n e (Fig. IC). Table II shows the d i s t r i b u t i o n of ~ a s e H a c t i v i t y in v a r i o u s rat tissues. A c t i v i t i e s of Mg ~-- and M n ~ ' - d e p e n d e n t RNases H w e r e h i g h in thymus, bone m a r r o w and spleen, w h i c h u n d e r g e high level
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
TABLE
1499
I
DNA s y n t h e s i s and levels of a c t i v i t i e s of RNA p o l y m e r a s e I and RNase H as a f u n c t i o n of time of d e p r i v a t i o n of protein, and s u p p l e m e n t a t i o n w i t h p r o t e i n and t h y r o i d h o r m o n e
Days on p r o t e i n -
RNA
free d i e t
polymerase
(nmol/mg
Mn 2+
DNA)
(pmol/mg
H
M g 2+
DNA)
DNA synthesis b
(cpm/mg
DNA)
0.40
+ 0.04
3500 + 400
1500 + 140
480 +
60
1
0.45
+ 0.05
3380 + 380
1800 + 185
490 +
62
3
0.72
+ 0.06
3660
+ 350
2820
+ 310
5
0.82
+ 0.08
3790 + 415
3110
+ 340
468 +
70
50% c a s e i n
and a d d i t i o n s + T3
15% c a s e i n T3
b,
I
RNase
0
Treatment
a,
Nuclear
to d i e t a
0.92
~ 0.i0
7240 ~ 805
4220 ~ 410
16650
~ 2100
0.86
+ 0.09
5620
+ 610
3700 + 405
5820 +
815
0.84
~ 0.i0
4130 ~ 440
3300 ~ 350
2540 ~
400
The rats w e r e fed for the i n d i c a t e d times p r o t e i n - f r e e mash. The p r e p a r a t i v e m a s h e s w e r e r e m o v e d at the end of 5 days, the rats w e r e i n j e c t e d s u b c u t a n e o u s l y w i t h 100 ug of t r i i o d o t h y r o n i n e (T3) and they w e r e then fed a meal of the p r o t e i n free m a s h c o n t a i n i n g c a s e i n for 20 h. D N A s y n t h e s i s was m e a s u r e d by the i n c o r p o r a t i o n of [3H]thymidine. Each rat w a s given 5 uCi of [ H ] - t h y m i d i n e in the tail v e i n at 60 m i n b e f o r e killing. The s p e c i f i c a c t i v i t i e s of n u c l e a r ~ ; A w e r e m e a s u r e d as d e s c r i b e d (16).
L i v e r n u c l e i w e r e i s o l a t e d and a s s a y e d as d e s c r i b e d in M A T E R I A L S A N D METHODS. R e a c t i o n m i x t u r e for RNase H contained, in a final vol. of 0.5 ml, 50 umol of T r i s - H C l (pH 8.0), 5 um~l of MgCl 2 or 0.2 umol of MnCI2, 1 umol of 2 - m e r c a p t o e t h a n o l , [ H] RNADNA h y b r i d (9,000 c p m per 150 pmol of 3 H - l a b e l e d nucleotide) and i0 ul of e n z y m e s o l u t i o n (about 20 ug of protein). A f t e r 15 m i n i n c u b a t i o n at 25°C the r e a c t i o n was s t o p p e d by the a d d i t i o n of 0.i ml of 0.i M E D T A (pH 7.0), 0.4 m g b o v i n e s e r u m a l b u m i n and 0.5 ml of cold 10% t r i c h l o r o a c e t i c acid. The r e a c t i o n m i x t u r e s w e r e c e n t r i f u g e d and the s u p e r n a t a n t was c o l l e c t e d and c o u n t e d in T r i t o n X - 1 0 0 / t o l u e n e s c i n t i l l a t i o n fluid. Each value is the m e a n of the r e s u l t s o b t a i n e d f r o m 4 to 5 rats and the s t a n d a r d deviations.
1500
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
2
4
|,
2
_
-
0
0
~
Nt/M~R
FIG. 1 Phosphocellulose column chromatography of RNase H of ~ i v e r nuclei from nutritionally-manipulation rats. All the rats were given either a meal of the protein-free mash supplemented with 50% casein (A), or a meal of the protein-free mash for 5 days (B). The rats given a protein-free diet for 5 days were given a subcutaneous injection of triiodothyronine (i00 ug) and the meal with 50% casein at the end of 5 days, and liver samples were taken at 20 h after manipulations (C). The nuclei derived from 9 g of liver were suspended in 5 ml of 0.15 M NaCI containing 5 mM 2-mercaptoethanol, and stirred gently with a magnetic stirrer for 20 min at 0"C. The nuclear extracts after centrifugatioh were dialyzed against 5 mM potassium phosphate buffer (pH 7.7), 5 mM 2-mercaptoethanol and 10% glycerol (Buffer A) for 5 h. The dialyzed nuclear extracts were loaded onto phosphocellulose (Pll) column (6 x 1.6 cm) that had been previously equilibrated with Buffer A. After washing the column with 2.5 vols. of the same buffer, the elution was carried out with a linear gradient of KCI from 0 to 0.8 M containing Buffer A. Fractions of 1.0 ml were collected and 50 ul aliquots were taken to determine both Mn 2+- and Mg2+-dependent RNases H activities. The reaction was studied under same assay conditions described in Table I. The salt concentration was determined by conductance measurements. O , Mn2+-dep endent RNase H; • , Mg 2+dependent RNase H; • , KCI concentration. of cellular replication. From these results, it is suggested that the activities of both RNases H may be related to the activity of DNA synthesis. DISCUSSION We have shown that at least two distinct RNase H activities,
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
1501
TABLE II Distribution of RNase H in various tissues from rat Tissues
RNase H activity Mg 2+
Mn 2+
(pmol/mg protein) Thymus
463 ~ 52
780 ~ 81
Bone marrow
371 + 31
490 + 37
Spleen
203 ~ 30
315 ! 42
Testis
194 + 22
274 + 31
Lung
193 ~ 20
260 ~ 28
Liver
133 + 20
218 + 24
Cerebellum
105 + 14
148 + 20
Hon-cerebellar part
90 ~ 12
137 ~ 21
Kidney
90 ~ i0
130 ~ 20
Heart
88 +
115 + 12
9
Rats were killed by decapitation and every tissue was homogenized with i0 vols. of 0.25 M sucrose, 0.1 M Tris-HC1 (pH 8.1), 3 mM CaCI2, 0.5 M KCI and 5 mM 2-mercaptoethanol and centrifuged at 105,000 x ~ for 60 min. All enzyme preparations were used after dialysis against 0.025 M Tris-HC1 (pH 7.7), 10% glycerol and 5 mM 2-mercaptoethanol for 3 h. The assay conditions were described in Table I. In every tissue, 100-200 ~g o~ protein was used as enzyme sources. Each value is the mean of the results from 3 to 4 rats and the standard deviations.
Mg 2+- and M n 2 + - d e p e n d e n t enzymes, are present in rat liver nuclei. They are distinguished from one another by their ionic requirement, molecular weight, sedimentation coefficient, optimal pH and sensitivity of -SH reagents (1). The amount of nucleolar RNA (10), the volume of nucleolar material per nucleus (7-9), and the activity of nucleolar RNA polymerase (2-4) all increased in the hepatocytes of rats deprived of protein. We now find that the activities of the Mn2+- and Mg 2+dependent RNases H are raised in the liver nuclei from rats in which h e p a t ~ DNA replication has been induced by a dietal manipulation. Mg ~ -dependent RNase H activity is also raised in liver nuclei in protein-deficient rats in which the activity of RNA polymerase I becomes doubled in the liver cells. A similar result was obtained from rat liver at 24 h after a single injection of thioacetamide; the induction of RNA polymerase I activity by thioacetamide is accompanied by a corresponding increase in the activity of ~e~eM~i~e~n~-~e~d~n~l~aseI~
~nM~+~:~e~tT~eI~
1502
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
is r e m a r k a b l y c o n s t a n t for the a c t i v i t i e s from the various tissues. This ratio also c h a n g e s w i t h timeafter p r o t e i n deprivation. These results suggest that Mn z+- and M g Z + - d e p e n d e n t RNases H serve d i f f e r ent p h y s i o l o g i c a l function. The m e c h a n i s m by w h i c h d e f i c i e n c i e s of p r o t e i n cause an increase in M g 2 + - d e p e n d e n t RNase H remains to be e l u c i d a t e d a l t h o u g h it seems likely that the RNase H s t i m u l a t e s RNA p o l y m e r a s e I activity to alter the c o n f o r m a t i o n of D N A as shown in yeast (18). Dezelee et al (18) have shown that RNase HI (Mr=48,000, an exonuclease) from y e a s t binds p r e f e r e n t i a l l y to s i n g l e - s t r a n d e d n u c l e i c acids to a l t e r the c o n f o r m a t i o n of d o u b l e - s t r a n d e d DNA, and that c o n c o m i t a n t w i t h D N A s t r u c t u r a l changes, a d r a s t i c stimu l a t i o n of t r a n s c r i p t i o n was o b s e r v e d w i t h yeast RNA polymerase. These results s u g g e s t e d the i n v o l v e m e n t of RNase HI in c h r o m a t i n s t r u c t u r e and function. To know the p r e c i s e function of Mg 2+and M n 2 + - d e p e n d e n t RNases H, f u r t h e r studies are n e c e s s a r y to assign a role in t r a n s c r i p t i o n as well as r e p l i c a t i o n t h r o u g h the p u r i f i c a t i o n s of these RNases H. ACKNOWLEDGEMENTS This i n v e s t i g a t i o n was s u p p o r t e d in part by a G r a n t - i n - A i d for C a n c e r R e s e a r c h f r o m the M i n i s t r y of Education, Science and Culture, Japan. The authors are g r a t e f u l to Mrs Mieko U n n o - Y a n o k u r a for e x c e l l e n t t e c h n i c a l assistance. REFERENCES I. Y. SAWAI, M. UNNO and K. TSUKADA, Biochem. Biophys. Res. Commun. 84 313-321 (1978) 2. P. M A N D E L and C. Q U I R I N - S T R I C K E R , Life Sci. 6 1 2 9 9 - 1 3 0 3 (1968) 3. C. S H A W and L. X. FILLIOS, J. Nutr. 9 6 327-336 (1968). 4. R. P. BAILEY, M. J. VROOMAN, Y. SAWAI, K. TSUKADA, J. SHORT, and I. LIEBERMAN, Proc. Natl. Acad. Sci. U.S.A. 7_~3 3201-3205 (1976) 5. J. SHORT, N. B. ARMSTRONG, R. ZEMEL and I. LIEBERMAN, Biochem. Biophys. Res. Commun. 5-6 430-437 (1973) 6. J. SHORT, M. B. ARMSTRONG, M. A. KOLYTSKY, R. A. MITCHELL, R. ZEMEL and I. LIEBERMAN, in: C o n t r o l of P r o l i f e r a t i o n in A n i m a l Cells (Clarkson, B. and Baserga, R. eds) pp. 37-48, C o l d S p r i n g H a r b o r L a b o r a t o r y , Cold Spring Harbor, N. Y. 7. R. E. STOWELL, C a n c e r 2 121-131 (1949). 8. U. STENRAM, Exp. Cell Res. 5 539-541 (1953). 9. D. S V O B O D A and J. HIGGINSON, Am. J. Pathol. 45 353-380 (1964). i0. U. STENRAM, Exp. Cell Res. 15 174-183 (1958). ii. W. E. LYNCH, R. F. BROWN, T. UMEDA, S. L A N G R E T H and I. L I E B E R M A ~ J. Biol. Chem. 245 3911-3916 (1970). 12. J. G. S T A V R I A N O P O U L O S , J. D. KARKAS and E. CHARGAFF, Proc. Natl. Acad. Sci. U.S.A. 6_~9 2 6 0 9 - 2 6 1 3 (1972). 13. Y. SAWAI and K. TSUKADA, Biochim. Biophys. A c t a 472 126-131 (1977). 14. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARRS and R. F. RANDALL, J. Biol. Chem. 193 265-275 (1951). 15. K. BURTON, Biochem. J. 61 473-483 (1955). 16. J. SHORT, K. TSUKADA, W. A. RUDERT and I. LIEBERMAN, J. Biol. Chem. 250 3602-3606 (1975). 17. K. TSUKADA, Y. SAWAI, J. SAITO and F. SATO, Biochem. Biophys.
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
Res. Con~nun. 8--52 8 0 - 2 8 6 (1978) 18. S. D E Z E L E E , F. W Y E R S , J-L, DARLIX, A. J. Biol. Chem. 252 8 9 3 5 - 8 9 4 4 (1977)
SENTENAC
1503
and P. F R O M A G E O T
Persamon Presl
CHANGE OF NUCLEAR RIBONUCLEASE H ACTIVITY FOLLOWING DEOXYRIBONUCLEIC ACID SYNTHESIS IN LIVER OF PROTEINDEFICIENT RAT Yaeuko Sawai, KenJi Wada and KinJi Tsukada Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo i01, Japan (Recelved In final form March 3, 1980) SUMMARY The activities of RNA polymerase I and Mg 2+dependent ribonuclease H (RNase H) become doubled in the liver nuclei of rats that are fed for several days a diet that lacks amino acids or protein. Both Mn 2+and Mg2+-dependent RNases H activities increased twoto three-~old in the liver nuclei of rats in which hepatic DNA replication has been induced by a dletal manipulation. Ribonucleaee H (RNaee H), which cleaves the RNA moiety of RNA-DNA hybrid molecules, is a ubiquitous enzyme in eukaryotlc, although the function of this enzyme still remains to be elucidated. A preceding report from our laboratory has shown that rat ~ liver contains at least two different RNames H a~ivities, an Me'-dependent RNase H of 150,000 daltons, and an Mg'~-dependent RName H of 35,000 daltons, distinguished from one another by physical and catalytic properties (i). The activity of nucleolar RNA polymorale increases in the hepatocytes of rats deprived of amino acids (2-4). In protein-deflcients, nuclear DNA synthesis and mitosis rasumm in some of the liver parenchymal cells soon after the animal is fed a meal containing protein (5) or some of the essential amino acids (6). The nucleolar changes including hypertrophy (79), an accumulation of RNA (10), and an increased activity of nucleolar RNA polymerase (2-4), characterize the liver of the amino acid-deficient rat as well as after partial hepatectomy. We have asked whether the activities of two nuclear RNasem H are elevated in the livers of rats induced to make DNA as well as RNA. This report describes that Mn2÷-dependent RNase H is involved in DNA synthesis, and Mg2÷-dependent RNase H is involved in synthesis of RNA and probably of DNA in liver nuclei. MATERIALS AND METHODS Male ~istar rats (140-150 g) were obtained locally. Unless otherwise specified, they were freely given protein-free diet and 50% casein to the time of the experiment. The protein-free diet was prepared by Teklad Mills, Madison Wisconsin, and consisted of 64% corn starch, 8% glucose, 10% corn oil, 4% "Salt Mix, U.S.A. XIV", 2% "Vitamin Fortification Mix" and 12% nonnutritive fiber. 002~-3205/80/181497-07$02.00/0 Copyrishc (c) 1980 PerSamon Prasm Lid
1498
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
Nuclei were i s o l a t e d from 10% h o m o g e n a t e of liver in s o l u t i o n c o n t a i n i n g only sucrose and CaCI 2 (ii). N u c l e i from 3 g of w e t liver were s u s p e n d e d in 1.5 ml of 0.15 M NaCl c o n t a i n i n g 5 m M 2m e r c a p t o e t h a n o l for 20 min at 2°C. The s u s p e n s i o n was c e n t r i f u g e d for 5 m i n at 10,000 x ~, the s u p e r n a t a n t fluid was a s s a y e d for RNase H a c t i v i t y and the p r e c i p i t a t e was used for assay of RNA p o l y m e r a s e I. RNA p o l y m e r a s e I was m e a s u r e d by its i n s e n s i b i l i t y of h i g h levels of ~ - a m a n i t i n w i t h liver nuclei by the m e t h o d s d e s c r i b e d p r e v i o u s l y (4). R a d i o a c t i v e l y l a b e l e d D N A - R N A h y b r i d was s y n t h e s i z e d on d e n a t u r e d calf thymus DNA using E.coli RNA p o l y m e r a s e as d e s c r i b e d p r e v i o u s l y (12). The assay of RNase H was based on the r e l e a s e into acid soluble fraction, of r a d i o a c t i v e m a t e r i a l from [3H]-RNA h y b r i d i z e d to DNA as d e s c r i b e d p r e v i o u s l y (13). P r o t e i n was d e t e r m i n e d DNA by the m e t h o d of B u r t o n
by the m e t h o d (15).
of Lowry
et al
(14)
and
RESULTS The s p e c i f i c a c t i v i t i e s of RNase H and RNA p o l y m e r a s e I in the n u c l e a r p r e p a r a t i o n s w e r e m e a s u r e d as a f u n c t i o n of time of d e p r i v a t i o n of amino acids. As shown in Table I, M g 2 + - d e p e n d e n t RNase H and RNA p o l y m e r a s e I rose g r a d u a l l y for several days. As c a l c u l a t e d from the Table, similar increase~ w e r e found in the a c t i v i t y of RNA p o l y m e r a s e I and that of M g 2 + - d e p e n d e n t RNase H, for the a c t i v i t y ratio of RNA p o l y m e r a s e I to M g 2 + - d e p e n d e n t RNase H r e m a i n e d c o n s t a n t for s e v e r a l days tested. Under these conditions, w h e n the e n z y m e a s s a y was p e r f o r m e d u s i n g Mn 2+, no significant d i f f e r e n c e in the level of RNase H a c t i v i t y was detected. A single m e a l c o n t a i n i n g p r o t e i n c a u s e d n u c l e a r DNA r e p l i c a t i o n in some of the h e p a t o c y t e s of the p r o t e i n - d e f i c i e n t rat (5, 6). As shown in Table I, s u p p l e m e n t a t i o n of the p r o t e i n - f r e e m a s h w i t h 50% casein and t h y r o i d h o r m o n e e f f e c t on the s u b s e q u e n t f o r m a t i o n of DNA, after 20 h. Under these conditions, b o t h Mg ~-- and Mn ~ d e p e n d e n t RNases H a c t i v i t i e s h a d i n c r e a s e d two- to t h r e e - f o l d w i t h i n 20 h (Table I). DNA f o r m a t i o n could be i n d u c e d in the liver of the p r o t e i n - d e f i c i e n t rat by feeding 15% c a s e i n Qr t h y r o i d h o r m o n e b u t the e f f e c t was smaller. The a c t i v i t i e s of Mg 2+- and M n 2 + - d e p e n d e n t RNases H w e r e r e d u c e d by such a treatment. Fig. 1 shows the p h o s p h o c e l l u l o s e c o l u m n c h r o m a t o g r a p h y Mn2+ p r o f i l e of n u c l e a r RNase H. Two m a j o r peaks c o r r e s p o n d i n g to and M g 2 + - d e p e n d e n t RNases H w e r e e l u t e d at a b o u t 0.2 M and 0.4 M KCI and a m i n o r p e a k h a v i n g RNase H a c t i v i t y was r e c o v e r e d in the f l o w - t h r o u g h f r o m2+ the column. As shown in" Fig. IB, the total activity of the Mg - d e p e n d e n t RNase H was about 2-fold h i g h e r at 5 days w i t h d e f i c i e n t rats, w h e r e a s the a c t i v i t y of M n 2 + - d e p e n d e D t RNase H w a s r e l a t i v e l y unaffected. However, both Mg 2+- and Mn 2+d e p e n d e n t RNases H a c t i v i t i e s w e r e i n c r e a s e d about 2- to 3-fold w h e n DNA s y n t h e s i s was induced by a meal of 50% c a s e i n and t h y r o i d h o r m o n e (Fig. IC). Table II shows the d i s t r i b u t i o n of ~ a s e H a c t i v i t y in v a r i o u s rat tissues. A c t i v i t i e s of Mg ~-- and M n ~ ' - d e p e n d e n t RNases H w e r e h i g h in thymus, bone m a r r o w and spleen, w h i c h u n d e r g e high level
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
TABLE
1499
I
DNA s y n t h e s i s and levels of a c t i v i t i e s of RNA p o l y m e r a s e I and RNase H as a f u n c t i o n of time of d e p r i v a t i o n of protein, and s u p p l e m e n t a t i o n w i t h p r o t e i n and t h y r o i d h o r m o n e
Days on p r o t e i n -
RNA
free d i e t
polymerase
(nmol/mg
Mn 2+
DNA)
(pmol/mg
H
M g 2+
DNA)
DNA synthesis b
(cpm/mg
DNA)
0.40
+ 0.04
3500 + 400
1500 + 140
480 +
60
1
0.45
+ 0.05
3380 + 380
1800 + 185
490 +
62
3
0.72
+ 0.06
3660
+ 350
2820
+ 310
5
0.82
+ 0.08
3790 + 415
3110
+ 340
468 +
70
50% c a s e i n
and a d d i t i o n s + T3
15% c a s e i n T3
b,
I
RNase
0
Treatment
a,
Nuclear
to d i e t a
0.92
~ 0.i0
7240 ~ 805
4220 ~ 410
16650
~ 2100
0.86
+ 0.09
5620
+ 610
3700 + 405
5820 +
815
0.84
~ 0.i0
4130 ~ 440
3300 ~ 350
2540 ~
400
The rats w e r e fed for the i n d i c a t e d times p r o t e i n - f r e e mash. The p r e p a r a t i v e m a s h e s w e r e r e m o v e d at the end of 5 days, the rats w e r e i n j e c t e d s u b c u t a n e o u s l y w i t h 100 ug of t r i i o d o t h y r o n i n e (T3) and they w e r e then fed a meal of the p r o t e i n free m a s h c o n t a i n i n g c a s e i n for 20 h. D N A s y n t h e s i s was m e a s u r e d by the i n c o r p o r a t i o n of [3H]thymidine. Each rat w a s given 5 uCi of [ H ] - t h y m i d i n e in the tail v e i n at 60 m i n b e f o r e killing. The s p e c i f i c a c t i v i t i e s of n u c l e a r ~ ; A w e r e m e a s u r e d as d e s c r i b e d (16).
L i v e r n u c l e i w e r e i s o l a t e d and a s s a y e d as d e s c r i b e d in M A T E R I A L S A N D METHODS. R e a c t i o n m i x t u r e for RNase H contained, in a final vol. of 0.5 ml, 50 umol of T r i s - H C l (pH 8.0), 5 um~l of MgCl 2 or 0.2 umol of MnCI2, 1 umol of 2 - m e r c a p t o e t h a n o l , [ H] RNADNA h y b r i d (9,000 c p m per 150 pmol of 3 H - l a b e l e d nucleotide) and i0 ul of e n z y m e s o l u t i o n (about 20 ug of protein). A f t e r 15 m i n i n c u b a t i o n at 25°C the r e a c t i o n was s t o p p e d by the a d d i t i o n of 0.i ml of 0.i M E D T A (pH 7.0), 0.4 m g b o v i n e s e r u m a l b u m i n and 0.5 ml of cold 10% t r i c h l o r o a c e t i c acid. The r e a c t i o n m i x t u r e s w e r e c e n t r i f u g e d and the s u p e r n a t a n t was c o l l e c t e d and c o u n t e d in T r i t o n X - 1 0 0 / t o l u e n e s c i n t i l l a t i o n fluid. Each value is the m e a n of the r e s u l t s o b t a i n e d f r o m 4 to 5 rats and the s t a n d a r d deviations.
1500
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
2
4
|,
2
_
-
0
0
~
Nt/M~R
FIG. 1 Phosphocellulose column chromatography of RNase H of ~ i v e r nuclei from nutritionally-manipulation rats. All the rats were given either a meal of the protein-free mash supplemented with 50% casein (A), or a meal of the protein-free mash for 5 days (B). The rats given a protein-free diet for 5 days were given a subcutaneous injection of triiodothyronine (i00 ug) and the meal with 50% casein at the end of 5 days, and liver samples were taken at 20 h after manipulations (C). The nuclei derived from 9 g of liver were suspended in 5 ml of 0.15 M NaCI containing 5 mM 2-mercaptoethanol, and stirred gently with a magnetic stirrer for 20 min at 0"C. The nuclear extracts after centrifugatioh were dialyzed against 5 mM potassium phosphate buffer (pH 7.7), 5 mM 2-mercaptoethanol and 10% glycerol (Buffer A) for 5 h. The dialyzed nuclear extracts were loaded onto phosphocellulose (Pll) column (6 x 1.6 cm) that had been previously equilibrated with Buffer A. After washing the column with 2.5 vols. of the same buffer, the elution was carried out with a linear gradient of KCI from 0 to 0.8 M containing Buffer A. Fractions of 1.0 ml were collected and 50 ul aliquots were taken to determine both Mn 2+- and Mg2+-dependent RNases H activities. The reaction was studied under same assay conditions described in Table I. The salt concentration was determined by conductance measurements. O , Mn2+-dep endent RNase H; • , Mg 2+dependent RNase H; • , KCI concentration. of cellular replication. From these results, it is suggested that the activities of both RNases H may be related to the activity of DNA synthesis. DISCUSSION We have shown that at least two distinct RNase H activities,
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
1501
TABLE II Distribution of RNase H in various tissues from rat Tissues
RNase H activity Mg 2+
Mn 2+
(pmol/mg protein) Thymus
463 ~ 52
780 ~ 81
Bone marrow
371 + 31
490 + 37
Spleen
203 ~ 30
315 ! 42
Testis
194 + 22
274 + 31
Lung
193 ~ 20
260 ~ 28
Liver
133 + 20
218 + 24
Cerebellum
105 + 14
148 + 20
Hon-cerebellar part
90 ~ 12
137 ~ 21
Kidney
90 ~ i0
130 ~ 20
Heart
88 +
115 + 12
9
Rats were killed by decapitation and every tissue was homogenized with i0 vols. of 0.25 M sucrose, 0.1 M Tris-HC1 (pH 8.1), 3 mM CaCI2, 0.5 M KCI and 5 mM 2-mercaptoethanol and centrifuged at 105,000 x ~ for 60 min. All enzyme preparations were used after dialysis against 0.025 M Tris-HC1 (pH 7.7), 10% glycerol and 5 mM 2-mercaptoethanol for 3 h. The assay conditions were described in Table I. In every tissue, 100-200 ~g o~ protein was used as enzyme sources. Each value is the mean of the results from 3 to 4 rats and the standard deviations.
Mg 2+- and M n 2 + - d e p e n d e n t enzymes, are present in rat liver nuclei. They are distinguished from one another by their ionic requirement, molecular weight, sedimentation coefficient, optimal pH and sensitivity of -SH reagents (1). The amount of nucleolar RNA (10), the volume of nucleolar material per nucleus (7-9), and the activity of nucleolar RNA polymerase (2-4) all increased in the hepatocytes of rats deprived of protein. We now find that the activities of the Mn2+- and Mg 2+dependent RNases H are raised in the liver nuclei from rats in which h e p a t ~ DNA replication has been induced by a dietal manipulation. Mg ~ -dependent RNase H activity is also raised in liver nuclei in protein-deficient rats in which the activity of RNA polymerase I becomes doubled in the liver cells. A similar result was obtained from rat liver at 24 h after a single injection of thioacetamide; the induction of RNA polymerase I activity by thioacetamide is accompanied by a corresponding increase in the activity of ~e~eM~i~e~n~-~e~d~n~l~aseI~
~nM~+~:~e~tT~eI~
1502
Liver Nuclear RNase H
Vol. 26, No. 18, 1980
is r e m a r k a b l y c o n s t a n t for the a c t i v i t i e s from the various tissues. This ratio also c h a n g e s w i t h timeafter p r o t e i n deprivation. These results suggest that Mn z+- and M g Z + - d e p e n d e n t RNases H serve d i f f e r ent p h y s i o l o g i c a l function. The m e c h a n i s m by w h i c h d e f i c i e n c i e s of p r o t e i n cause an increase in M g 2 + - d e p e n d e n t RNase H remains to be e l u c i d a t e d a l t h o u g h it seems likely that the RNase H s t i m u l a t e s RNA p o l y m e r a s e I activity to alter the c o n f o r m a t i o n of D N A as shown in yeast (18). Dezelee et al (18) have shown that RNase HI (Mr=48,000, an exonuclease) from y e a s t binds p r e f e r e n t i a l l y to s i n g l e - s t r a n d e d n u c l e i c acids to a l t e r the c o n f o r m a t i o n of d o u b l e - s t r a n d e d DNA, and that c o n c o m i t a n t w i t h D N A s t r u c t u r a l changes, a d r a s t i c stimu l a t i o n of t r a n s c r i p t i o n was o b s e r v e d w i t h yeast RNA polymerase. These results s u g g e s t e d the i n v o l v e m e n t of RNase HI in c h r o m a t i n s t r u c t u r e and function. To know the p r e c i s e function of Mg 2+and M n 2 + - d e p e n d e n t RNases H, f u r t h e r studies are n e c e s s a r y to assign a role in t r a n s c r i p t i o n as well as r e p l i c a t i o n t h r o u g h the p u r i f i c a t i o n s of these RNases H. ACKNOWLEDGEMENTS This i n v e s t i g a t i o n was s u p p o r t e d in part by a G r a n t - i n - A i d for C a n c e r R e s e a r c h f r o m the M i n i s t r y of Education, Science and Culture, Japan. The authors are g r a t e f u l to Mrs Mieko U n n o - Y a n o k u r a for e x c e l l e n t t e c h n i c a l assistance. REFERENCES I. Y. SAWAI, M. UNNO and K. TSUKADA, Biochem. Biophys. Res. Commun. 84 313-321 (1978) 2. P. M A N D E L and C. Q U I R I N - S T R I C K E R , Life Sci. 6 1 2 9 9 - 1 3 0 3 (1968) 3. C. S H A W and L. X. FILLIOS, J. Nutr. 9 6 327-336 (1968). 4. R. P. BAILEY, M. J. VROOMAN, Y. SAWAI, K. TSUKADA, J. SHORT, and I. LIEBERMAN, Proc. Natl. Acad. Sci. U.S.A. 7_~3 3201-3205 (1976) 5. J. SHORT, N. B. ARMSTRONG, R. ZEMEL and I. LIEBERMAN, Biochem. Biophys. Res. Commun. 5-6 430-437 (1973) 6. J. SHORT, M. B. ARMSTRONG, M. A. KOLYTSKY, R. A. MITCHELL, R. ZEMEL and I. LIEBERMAN, in: C o n t r o l of P r o l i f e r a t i o n in A n i m a l Cells (Clarkson, B. and Baserga, R. eds) pp. 37-48, C o l d S p r i n g H a r b o r L a b o r a t o r y , Cold Spring Harbor, N. Y. 7. R. E. STOWELL, C a n c e r 2 121-131 (1949). 8. U. STENRAM, Exp. Cell Res. 5 539-541 (1953). 9. D. S V O B O D A and J. HIGGINSON, Am. J. Pathol. 45 353-380 (1964). i0. U. STENRAM, Exp. Cell Res. 15 174-183 (1958). ii. W. E. LYNCH, R. F. BROWN, T. UMEDA, S. L A N G R E T H and I. L I E B E R M A ~ J. Biol. Chem. 245 3911-3916 (1970). 12. J. G. S T A V R I A N O P O U L O S , J. D. KARKAS and E. CHARGAFF, Proc. Natl. Acad. Sci. U.S.A. 6_~9 2 6 0 9 - 2 6 1 3 (1972). 13. Y. SAWAI and K. TSUKADA, Biochim. Biophys. A c t a 472 126-131 (1977). 14. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARRS and R. F. RANDALL, J. Biol. Chem. 193 265-275 (1951). 15. K. BURTON, Biochem. J. 61 473-483 (1955). 16. J. SHORT, K. TSUKADA, W. A. RUDERT and I. LIEBERMAN, J. Biol. Chem. 250 3602-3606 (1975). 17. K. TSUKADA, Y. SAWAI, J. SAITO and F. SATO, Biochem. Biophys.
Vol. 26, No. 18, 1980
Liver Nuclear RNase H
Res. Con~nun. 8--52 8 0 - 2 8 6 (1978) 18. S. D E Z E L E E , F. W Y E R S , J-L, DARLIX, A. J. Biol. Chem. 252 8 9 3 5 - 8 9 4 4 (1977)
SENTENAC
1503
and P. F R O M A G E O T