- Email: [email protected]
DNA biosynthesis in nuclei
216
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97380
DNA BIOSYNTHESIS IN NUCLEI I. CHARACTERIZATION OF DNA SYNTHESIS BY ISOLATED RAT L I V E R NUCLEI USING ENDOGENOUS DNA AS P R I M E R ~ G R E G O R Y S. P R O B S T , E L I Z A B E T H B I K O F F * * , S T E P H E N J. K E L L E R AND R A L P H R. MEYER
Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 4522r (U.S. 4.) (Received May 8th, 1972)
SUMMARY
I. A DNA biosynthetic system consisting of isolated nuclei using endogenous DNA as primer has been characterized. The nuclei are capable of incorporating E3HI d T T P into DNA linearly for 2 h, and incorporation is proportional to protein concentration up to 2 mg/ml. 2. Preincubation at 37 °C greatly enhances incorporation, but heating for 4 min at 65 °C destroys activity. The pH optimum is 8.0 with Tris-HC1 and 9.0 with glycine-NaOH buffer. The system requires Mg2÷ and is stimulated by ATP, KC1, sucrose and glycerol; but deoxyribonuclease, polyamines, Na~P207, actinomycin D, ethidium bromide and acriflavin inhibit incorporation. 3. Addition of native DNA to the reaction stimulates incorporation 5-told. DNA synthesis by endogenously and exogenously primed nuclei has been compared. CsCI density gradient centrifugation indicates that endogenously primed reactions synthesize rat liver DNA, but addition of exogenous DNA causes inhibition of endogenous DNA synthesis in favor of the exogenous DNA.
INTRODUCTION
In recent years considerable interest has been focused on the mechanism of DNA replication in the eukaryotic chromosome. While an ever-increasing number of studies of crude and purified DNA polymerase have appeared z-n, there have been surprisingly few studies of DNA synthesis by isolated nuclei. The use of intact nuclei offers the advantage of an in vitro system which can be manipulated while at the same time provides a native state at the site of replication which closely approximates that in vivo. While a few reports using nuclear systems have been published 1~-19, in most cases exogenous DNA has been added. Thus, it can be questioned whether the Abbreviation: BBOT, 2,5-bis(2-(tert-butylbenzoxaxolyl)) thiophene. * A p r e l i m i n a r y r e p o r t of p a r t of this w o r k h a s been published 1. The D N A required for enzymatic activity is referred to as the " p r i m e r " in a general sense since its chemical relationship to the p r o d u c t has not been established. ** P r e s e n t address: D e p a r t m e n t of Biological Sciences, Columbia University, New York, N.Y., lOO2 7.
Biochim. Biophys. Acta, 281 (1972) 216-227
DNA BIOSYNTHESISIN NUCLEI. I
217
characteristics of these systems accurately reflect those of the DNA polymerase in its native state, or those of a crude enzyme extract consisting of solubilized DNA polymerase acting on the exogenous DNA. Furthermore, recent studies have indicated that the DNA polymerase of Escheriehia coli isolated by Kornberg ~° and long considered the DNA replicating enzyme, may actually function in DNA repair 2J, and that there are at least two other DNA polymerases present in E. coli ~ presumably one of which is involved in DNA replication. This suggests an advantage of using whole organelles to synthesize DNA: under these conditions one is more likely (although it is not absolutely certain) to be dealing with the DNA replicating system rather than a DNA repair system. This rationale has also led to the use of toluenized cells to study DNA replication in E. coli 38,2, As part of a continuing study of DNA replication in eukaryotes 25-2~, the present study was undertaken (a) to characterize a DNA biosynthetic system from intact nuclei which uses endogenous DNA as a primer, (b) to compare DNA synthesis in nuclei primed by endogenous and exogenous DNA, (c) to serve as a basis for comparison to DNA synthesis by purified nuclear DNA polymerase, and (d) to serve as a basis for comparison" to DNA synthesis by isolated mitochondria 29,3°.
EXPERIMENTAL PROCEDURE
Matermls Unlabeled deoxyribonucleoside triphosphates, ATP, p-nitrophenyl phosphate, and neutral acriflavin were purchased from Sigma Chemical Co., St. Louis, Mo. Thymidine-5'-monophospho-p-nitrophenyl ester, thymidine-3'-monophospho-p-nitrophenyl ester, spermine, spermidine, pronase A and ethidium bromide were obtained from Calbiochem, Los Angeles, Calif. Actinomycin D and bovine serum albumin were purchased from Mann Research Labs, New York, N. Y., while calf thymus DNA, ribonucleases A and T 1 and deoxyribonuclease were obtained from Worthington Biochemical Corp., Freehold, N. J. CsC1 (optical grade) was a product of Gallard and Schlesinger Chemical Co., Carle Place, N. Y. and Micrococcus lysodeikticus and Clostridium per]ringens DNA was a product of Miles Laboratories, Elkhart, Ind. I3HIdTTP, E3H~dATP, I~HIdGTP, and [*HJthymine were obtained from New England Nuclear Corp., Boston, Mass., and E3HIdATP and [SHIdCTP from Schwarz BioResearch, Orangeburg, N. Y. Radioisotopes supplied in 5 ° % ethanol were used directly without removal of the ethanol. Radioactive DNA was prepared from E. coli strain 15 T - H - U - using ~SHJthymine, and activated DNA was prepared as described previously 2~. Denatured DNA was prepared by heating samples at IOO °C for 15 rain followed by rapid cooling. Methods Enzyme assays. DNA polymerase assays were carried out in 12 mm × 75 mm glass tubes in a final reaction volume of 125/A. Assays components are given in the legends. After incubation, IOO/A samples were spotted on filter paper discs, precipitated in io % trichloroacetic acid, and processed for liquid scintillation counting in " Details of these e x p e r i m e n t s will be published in a later c o m m u n i c a t i o n . A preliminary r e p o r t has been published 28.
Biochim. Biophys. Acta, 281 (1972) 216-227
218
G.S. PROBST et al.
2,5-bis-(2-(tert-butylbenzoxaxolyl)) thiophene (BBOT)-toluene as described previously 31. Assays for acid phosphatase, alkaline phosphatase, acid phosphodiesterase I, alkaline phosphodiesterase I, phosphodiesterase II, deoxyribonuclease I and II were performed as before 2~. Isolation of nuclei. In order to obtain large quantities of nuclei, the Blobel and Potter 32 method was scaled up and modified. In a typical preparation 9° g of liver from rats weighing 175-200 g were homogenized with a Potter-Elvehjem homogenizer in a final volume of 270 ml of 0.25 M sucrose in Buffer A (0.05 M Tris-HC1 buffer (pH 8.o), 0.025 M KC1, 0.005 M 2-mercaptoethanol, o.ooi M EDTA, and 0.005 M magnesiumacetate). In some experiments 0.005 M CaC12 was substituted for magnesiumacetate ("calcium nuclei"). The homogenate was passed through four layers of cheesecloth and centrifuged at 800 x g for IO rain. The nuclear pellets were washed twice and suspended in 45 ml of 0.25 M sucrose Buffer A to which was then added 9 ° ml of a 2. 3 M sucrose solution made up in Buffer A. After thorough mixing, 45 ml of this suspension was overlaid onto a 15 ml cushion of 2. 3 M sucrose in Buffer A in each of the tubes. Centrifugation was carried out in a Spinco SW-25.2 rotor at 25 ooo rev./min for 2 h. The pellets were taken up in 20 ml of Buffer A containing 20 *I,, glycerol and frozen in liquid N 2 or at 20 °C. The yield ~f ruclei by this method was about 7 ° °'o as estimated from the recovery of DNA from the filtered homogenate. CsC1 centrifugation. A standard reaction mixture was scaled up to 4.o ml, IZHIdATP was used as a label in addition to [~HIdTTP, and incubation was carried out for 9 ° rain. The nuclei were lysed in 1. 7 % sodium dodecyl sulfate and the DNA extracted by a modified method of Kirby z3. The crude DNA was spooled from ethanol and dried over N 2. The DNA was dissolved in 0.03 M NaC1 0.o03 M sodium citrate, treated with 20/~g/ml ribonuclease A and 50 units/ml of ribonuclease T 1 for 3° rain at 37 °C, re-extracted with phenol, spooled from ethanol, dried over N 2 and dissolved again in 0.03 M NaCl-o.oo 3 M sodium citrate. Samples of 5 °/~g of DNA in CsC1 were run in the Spinco SW 5oL rotor for 60 h, using a "relaxed gradient TM. Five drop fractions were collected, diluted to I ml and the absorbance at 260 nm determined. Carrier DNA (75/~g) was added to the samples which were then spotted onto filter paper discs and prepared for liquid scintillation counting. Densities were determined with an Abbe refractometer. Other procedures. Protein concentrations were measured by the Lowry reaction using bovine serum albumin as a standard and DNA by the diphenylamine procedure ~6 using calf thymus DNA as a standard.
RESULTS
Nuclear purity and stability The purity of the preparations was checked by phase contrast and election microscopy. The modified Blobel and Potter method routinely yielded large quantities of nuclei free of red blood cells, whole liver cells and mitochondria. The nuclei as isolated are very stable when stored in liquid N 2 in buffers containing 20 O//oglycerol or sucrose. Nuclei incubated at 37 °C from 0.5-6 h prior to assay showed a marked increase Biochim. Biophys. Acta, 281 (1972) 216-227
DNA
219
BIOSYNTHESIS IN NUCLEI. I
in the ability to incorporate [3H]dTTP (Fig. I). Maximal stimulation (4.5-fold) was noted for the endogenous system with preincubation for 2 h at 37 °C. Similar results were found with the exogenously primed system, showing a 1.4-fold maximal increase in incorporation after 1-2 h of preincubation, and essentially no loss of maximal activity up to 6 h of preincubation (Fig. I). In contrast, 4 min incubation at 55 °C resulted in 5 ° % loss of activity and at 60 °C all activity was lost. I
I
I
I
I
I 3o
400
I
I
I
2s
300
o oc
c= 20C
,..
15
d
10C
1
I
I
I
I
J
I
0
1
2
3
4
5
6
HOURS
I
6O
I
120
I
1110
240
MINUTE~
Fig. I. E f f e c t of p r e i n c u b a t i o n of nuclei on n u c l e a r D N A s y n t h e s i s . Nuclei s t o c k w a s i n c u b a t e d a t 37 °C for t h e t i m e s i n d i c a t e d in t h e figure. S a m p l e s of 25/~1 were w i t h d r a w n a n d a s s a y e d for D N A p o l y m e r a s e a c t i v i t y for i h. T h e s t a n d a r d r e a c t i o n m i x c o n t a i n e d t h e following c o m p o n e n t s in a final v o l u m e of 125 ~1:o.o25 M Tris-HC1 b u f f e r (pH 8.o), 0.0o 5 M 2 - m e r c a p t o e t h a n o l , o.oo 5 M E D T A , 0.025 M KC], o.oi M m a g n e s i u m acetate, o.oo2 M A T P , o.o15 m M each of d A T P , d C T P , d G T P , a n d [ 3 H ] d T T P (specific a c t i v i t y , 1.41 Ci/mmole), a n d 15 % glycerol. W h e n e x o g e n o u s p r i m e r w a s used, n a t i v e calf t h y m u s D N A w a s a d d e d a t IOO ~ug/ml. O - O , w i t h e x o g e n o u s p r i m e r ; 0-0, with endogenous primer; ~, represents O and • simultaneously. Each assay contained i II lug of n u c l e a r protein. Fig. 2. I n c o r p o r a t i o n of E3H]dTTP into D N A b y isolated nucei over a 4-h period. T h e s t a n d a r d r e a c t i o n m i x t u r e as d e s c r i b e d in t h e legend to Fig. i w a s scaled u p to 4 m], a n d triplicate ioo/~1 s a m p l e s were w i t h d r a w n a t t h e t i m e s i n d i c a t e d in t h e figure. T h e r e a c t i o n m i x t u r e c o n t a i n e d 984 l, gllnl of n u c l e a r protein. O O , w i t h e x o g e n o u s p r i m e r ; 0 - • , with endogenous primer.
E//ect o/concentration o/nuclei Incorporation of labeled substrate into DNA was proportional to the amount of nuclei added up to approx. 2 mg/ml of protein (400/,g/assay).
Time course o/incorporation Incorporation of [3H]dTTP into DNA was fairly linear for a period of at least 2 h in nuclei primed with endogenous DNA and 1.5 h with exogenous DNA (Fig. 2). J Longer incubation led to a loss of labeled product. One of the malor advantages of our system is the ability to incorporate labeled substrate for long periods of time (2 h). Others using endogenously primed nuclear systems have obtained linear Biochim. Biophys. Acta, 281 (1972) 216-227
G . S . PROBST et al.
220
incorporation for only 5-4 o min 12'14'1s or have obtained very low levels of incorporation. We obtained I6~ pmoles/mg protein compared with 2.2-52.8 pmoles/mg of protein reported by others under normal conditions. We can obtain 8o5 pmoles/mg of protein with preincubated nuclei.
E//ect o~ exogenous DNA Addition of calf thymus DNA stimulates the reaction several fold which is consistent with previous studies 16-19. The degree of stimulation with DNA from several sources shows considerable variation (Table I). Native DNA was more effective than denatured DNA (Table II). Enzyme saturation is reached at a concentration of TABLE I ACTIVITY OF RAT L1VER NUCLEI USING VARIOUS D N A PRIMERS R e a c t i o n m i x t u r e s c o n t a i n e d t h e s t a n d a r d a s s a y c o m p o n e n t s as d e s c r i b e d in t h e l e g e n d to Fig. for e x o g e n o u s l y p r i m e d r e a c t i o n s e x c e p t t h a t each t u b e c o n t a i n e d t h e t y p e of D N A as g i v e n in t h e t a b l e , a n d i n c u b a t i o n w a s c a r r i e d o u t for I h. E a c h t u b e c o n t a i n e d 57.6/~g of n u c l e a r prot e i n. " A c t i v a t e d " D N A was p r e p a r e d b y l i m i t e d d e o x y r i b o n u c l e a s e digestion~L
Type of DNA
Biological source (/ DNA
Incorporation of [~H]dT TP pmoles %
Mammalian
Calf t h y m u s " A c t i v a t e d " calf t h y m u s R a t l i v e r nuclei Novikoff hepatoma
i o.o 19.2 6.83 9.82
too 192 68. 3 98.2
Algal
Euglena gracilis Chlamydomonas reinhardii E. coli M. lysodeikticus C. perfringens
3.89 2.81
38.9 28.1
6.78 5.78 8.78
67.8 57.8 87.8
Bacterial
TABLE
II
DNA SYNTHESIS The c o m p l e t e a s s a y m i x t u r e w a s as g i v e n in t h e l e g e n d t o Fig. i, e x c e p t t h a t t h e i n c u b a t i o n t i m e was I h. O m i s s i o n s or a d d i t i o n s t o t h i s were as g i v e n in t h e Table. E a c h a s s a y t u b e c o n t a i n e d 115 to 12o/~g of n u c l e a r p r o t e i n . F o r t h e e n d o g e n o u s p r i m e d r e a c t i o n i o o % a c t i v i t y w a s equiv a l e n t to 1.5-5.I p m o l e s a n d for t h e e x o g e n o u s p r i m e d r e a c t i o n 8.6-13.2 pmoles. SOME REQUIREMENTS FOR
System
Complete -- A T P KC1 - - M g ~+ -- g l y c e r o l --dATP -- d C T P --dGTP - - d A T P , dCTP, d G T P mercaptoethanol --EDTA +spermine (25o/~M) (375/~M) + s p e r m i d i n e (250/tM) (375 #M) --native DNA+denatured -
-
-
-
DNA
Activity (%) Endogenous primer
Exogenous primer
IOO 8 79 5 13 82 7o 47 38 99 98 9 5 4° 32 --
I oo 5 84 ~a 21 76 83 55 55 99 99 4° lo 79 79 37
Biochim. Biophys. Acta, 28I (1972) 216-227
DNA
BIOSYNTHESIS IN NUCLEI. I
221
about 2oo/~g/ml using native DNA but only 75/,g/ml for denatured DNA (Table II). Further increases in DNA concentration begin to inhibit DNA synthesis.
pH e//ects The effects of pH over the range 6.o-11.o using several different buffers were studied. With endogenously primed nuclei, Tris-HC1 or glycine-NaOH buffers were equally effective, although the pH optimum was considerably different (pH 8.0 and 9.0, respectively). With exogenously primed nuclei, the pH optima were approximately the same as with endogenously primed nuclei; however, the activity was almost 2-fold greater with Tris-HC1 than with glycine-NaOH buffer.
E/[ects o/divalent cations Mg~+ is required for DNA synthesis and has an optimum at 5-15 mM (Table II). An absolute requirement could not be demonstrated in this experiment because the nuclei are suspended in a MgS+-containing buffer. An absolute requirement could be demonstrated using Ca 2+ nuclei. Mn 2+ was only lO-29 % effective as Mg2+ at an optimum of o.5-1. 5 raM, and Ca 2+ was ineffective. However, Ca2+-isolated nuclei were consistently more active than Mg2+-isolated nuclei and produced different Mg2+ optima for endogenously and exogenously primed reactions.
E//ect o~ glycerol and sucrose
(~
Glycerol and sucrose stimulated incorporation of labeled substrate into DNA (Fig. 3). For the endogenous system, a 6-fold stimulation was obtained with 25 %
20
i
I
I
I
I
I
I
I
I
I
I
I
is
Z 0
o
t
I
I
I
I
1
5
lo
15
2o
25
3o
MINUTES
Fig. 3- The effects of glycerol and sucrose on D N A synthesis b y isolated nuclei. N u c l e i w e r e washed 3 times in Buffer A prior to use. The a s s a y m i x t u r e w a s t h e same as described in the legend to Fig. I except i n c u b a t i o n was for i h a n d glycerol was excluded. E i t h e r glycerol or sucrose w a s added to t h e individual reaction t u b e s to o b t a i n the c o n c e n t r a t i o n s indicated in the figure, a n d each reaction contained 12o p g of nuclear protein. /x_/x, sucrose w i t h exogenous p r i m e r ; A - A , sucrose w i t h endogenous primer; 0 - 0 , glycerol w i t h exogenous primer; Q - O , glycerol w i t h endogenous primer.
Biochim. Biophys. Acta, 281 (1972) 216-227
222
G . S . PROBST et al.
glycerol, and 7-fold with 3 ° % sucrose. Due to pipetting difficulties, concentrations above 3 ° o/jo could not be tested; thus, no optimum was found for the endogenous system with sucrose. With exogenous primer, glycerol gave a 4-fold stimulation at 15 %, and sucrose gave a 7-fold increase at 2 5 %. The role of sucrose or glycerol in promoting this stimulation is unknown. Whether it acts as a stabilizer of the enzymes involved in replication or if it plays a more central role remains to be determined.
E]/ect o/KCl Monovalent cations have been shown to be moderately stimulatory with most mammalian DNA polymerases studied ~v and highly stimulatory in the case of rat liver 25'27 and calf liver mitochondrial DNA polymerases (R. R. Meyer, unpublished data). Using rat liver nuclei, KC1 was found to stimulate to the extent of up to 2-fold at concentrations of 0.025-0.050 M (Table II). This stimulation is largely an ionic strength effect since other salts could be substituted for KC1. Some specific ion effect was observed since NH4C1 was considerably less effective than KC1 or NaC1.
Deoxyribonucleoside triphosphate requirement The effect of excluding one or more deoxyribonucleoside triphosphates is shown in Table II. Considerable activity is obtained which m a y be due to the presence of a deoxyribonucleoside triphosphate pool or the formation of small amounts of them by nucleoside kinases 14. There m a y be also limited addition of the labeled nucleoside triphosphates to the ends of DNA chains or there m a y be a terminal addition enzyme. present 3s-42. If a terminal addition enzyme is present, it can be questioned to what extent the incorporation in the presence of all four deoxyribonucleoside triphosphates represents DNA polymerase activity and to what extent it represents terminal addition. We feel we are measuring DNA polymerase for the most part, since terminal addition enzymes are primarily poly(A) polymerases 42, and there is recent evidence that terminal addition enzyme m a y be unique to calf thymus 43. Also, deoxyribonucleoside triphosphates inhibit the purified calf terminal addition enzymes which have been studied nv.41. Incorporation can be inhibited 80 % with actinomycin D (see below), and this antibiotic does not inhibit terminal addition enzymes '~.
A TP requirement Isolated nuclei are stimulated by ATP (Table II), with an optimum at 2- 3 mM. Similar effects of ATP have been noted for other nuclear systems 15'1v as well as partially purified DNA polymerases ~5,27. The role of ATP probably lies in inhibiting deoxyribonucleoside triphosphatases or effecting regeneration ot the triphosphate. No purified DNA polymerase has been found to require ATP, and in the case of rat liver mitochondrial DNA polymerase, ATP was no longer required after removal of a deoxyguanosine triphosphatase during purification 27.
E]/ect o/polyamines In the isolated nuclei of Physarum polycephalumxg, spermine greatly stimulates DNA synthesis. In the rat liver nuclei system, polyamines inhibit DNA synthesis (Table II). Brewer and Rusch 19 suggest that spermine promotes penetration of the exogenous primer into the nucleus or activates latent polymerase in the nucleus. Our results suggest that this effect m a y be species specific. Biochim. Biophys. Acta, 281 (1972) 216-227
DNA
BIOSYNTHESIS IN NUCLEI. I
223
Effect o/inhibitors The isolated nuclear system is sentitive to several known inhibitors of DNA polymerase as shown in Table I I I . Addition of sodium pyrophosphate, deoxyribonuclease I, actinomycin D, ethidium bromide or acriflavin markedly decrease activity, although ribonuclease and pronase have virtually no effect. The results of several treatments on the product of the reaction is also given. Treatment of the acid-precipitable product with hot trichloroacetic acid or deoxyribonuclease leads to almost complete solubilization of the product. Ribonuclease and pronase are ineffective. These studies serve to identify the product of the reaction as DNA. TABLE III EFFECTS OF ADDITION OF VARIOUS SUBSTANCES ON D N A SYNTHESIS BY RAT LIVER NUCLEI T h e c o n t r o l t u b e s c o n t a i n e d t h e s t a n d a r d a s s a y c o m p o n e n t s as d e s c r i b e d in t h e l e g e n d t o Fig. I e x c e p t t h a t t h e c o n c e n t r a t i o n of p r o t e i n w a s 3 o 0 / , g / t u b e , a d d i t i o n s were m a d e as g i v e n in t h e t a b l e a n d i n c u b a t i o n w a s for I h. I n E x p t B, a f t e r i n c u b a t i o n , t h e t u b e s w e re h e a t e d to 65 °C for 4 rain a n d cooled to i n a c t i v a t e t h e e n z y m e s . A d d i t i o n s as l i s t e d in t h e t a b l e w e re m a d e a n d i n c u b a t i o n c o n t i n u e d for i h a t 37 °C e x c e p t for t h e t r i c h l o r o a c e t i c a c i d t u b e s w h i c h were h e a t e d a t 90 °C for i o min.
Experiment
(A) a d d i t i o n s before i n c u b a t i o n Control I +deoxyribonuclease (80/tg/ml) + r i b o n u c l e a s e (80 p g / m l ) + N a ~ P z O 7 (4 ° mM) + e t h i d i u m b r o m i d e (500/~M) + a c r i f l a v i n (500/*M) + a c t i n o m y c i n D ~5oo/iM) (B) A d d i t i o n s a f t e r i n c u b a t i o n Control II + d e o x y r i b o n u c l e a s e (8o/~g/ml) + r i b o n u c l e a s e (80/~g/ml) + p r o n a s e (80/~g/ml) + t r i c h l o r o a c e t i c a c i d ( i o %)
Endogenous primer
Exogenous primer
[aH]dTTP incorporated (pmoles )
% control activity
[3H]dTTP incorporated (pmoles )
% control activity
IOO,O
21.o
IOO.O
o 8.5 0.03 o-76 2-44 2.17
8. 9
o 95.5 0. 3 8.5 27.4 24.3
o.15 21.9 0.06 5-7 lO-3 6.5
0. 7 lO4.2 0.2 27.1 49.0 3o.9
9.8
IOO.O
23. 9
IOO.O
o.oi
9.6 i3. 3 0.22
O.I
97.9 135. 7 2.2
0.25
24.0 24.2 o.o 7
I.O
lOO.4 i o i .2 0.2
Additional enzyme activities in nuclei Since phosphatases and nucleases have been shown to affect DNA synthesis n,20,27,~, nuclei were tested for the presence of these enzymes. The results, summarized in Table IV, indicate that nuclei contain phosphatases, phosphodiesterases and nucleases. In addition to these enzymes, nucleoside kinase, and nucleoside diphosphate kinase have been detected b y others 14, as well as a neutral protease in calf thymus nuclei 45. Thus, the role of these enzymes must be taken into consideration with interpreting the results of E3HJdTTP incorporation experiments.
Nature o[ the synthesized product Since the addition of native DNA can increase incorporation over 5-fold, the question arises as to the mechanism of this stimulation. There are several possibilities including (a) the exogenous DNA stimulates endogenous DNA synthesis, (b) exoBiochim. Biophys. Acta, 281 (1972) 216-227
224
G.S. PROBST et al.
TABLE IV ADDITIONAL
ENZYME
ACTIVITIES
IN ISOLATED
RAT LIVER
NUCLEI
Pbosphomonoesterase, phosphodiesterase and deoxyribonuclease activities were determined as described previously2s. Each assay tube contained 222/tg of nuclear protein. For the esterase i unit of activity is equivalent to the release of i pnlole of p-nitrophenol/h at 37 °C. For deoxyribonuclease I unit of activity is equivalent to the release of i nmole of nucleotide per 3° min at 37 °C. Enzyme
Activity (units/rag protein)
Alkaline phosphatase Acid phosphatase Alkaline phosphodieterase 1 Acid phosphodiesterase I Phosphodiesterase II
0.044 0.293 o.o73 0.038 o.oi9
Acid deoxyribonuclease native DNA substrate denatured DNA substrate Alkaline deoxyribonuclease native DNA substrate denatured DNA substrate
14.8 I33.o 12. 4 I O I .O
genous D N A is being s y n t h e s i z e d outside of t he nucleus b y solubilized D N A polymerase, or (c) t h e exogenous D N A enters th e nucleus and is synthesized b y t h e endogenous D N A polymerase. To test these possibilities, reactions were scaled up, an d after i n c u b a t i o n , th e d i s t r i b u t i o n of r a d i o a c t i v e label b et w een the s u p e r n a t a n t and t h e pellet was d e t e r m i n e d . In a n o t h e r set of tubes, E3HIDNA was a d d e d as p r i m e r a nd I 3 H I d T T P was replaced b y u n l a b e l e d d T T P . These tubes were i n c u b a t e d an d t h e n s e p a r a t e d into s u p e r n a t a n t a n d pellet fractions. Th e results of these e x p e r i m e n t s are shown in Tab l e V. T h e d a t a suggest t h a t w i t h endogenous primer almost all of the synthesis occurs in t h e nucleus. T h e 5 % of r a d i o a c t i v i t y found in t h e s u p e r n a t a n t m a y be due to f r a g m e n t e d nuclei. W i t h e x o g e no u sl y p r i m e d reaction, only 20 o//o of t h e label was f o u n d outside of t h e nucleus, p r e s u m a b l y due to D N A polymerase w hich h a d leached out. T h e bulk of t h e label, however, is associated w i t h t h e nucleus. T h e results of the [ 3 H I D N A p r i m e d e x p e r i m e n t s clearly show t h a t g r e a t e r t h a n 7 ° % of the p r i m e r D N A binds to th e nucleus, i n d i c a t i n g t h a t the bulk of the synthesis is
TABLE V DISTRIBUTIONOF RADIOACTIVEDNA BETWEEN SUPERNATANTAND NUCLEARPELLET Exogenous and endogenous reactions (A and B) were run as described in the legend to Fig. i except that they were scaled up to i ml, 1.14 mg of nuclear protein was used and incubation time was I h. At the end of the incubation the tubes were centrifuged at 800 × g to pellet the nuclei, and the distribution of radioactivity between the supernatant and pellet determined. In a third series of tubes (C) [SHIDNA was added, [aH]dTTP omitted and the distribution of label determined after incubation Experimental condition
(A) Exogenous primer (B) Endogenous primer (C) ?H]DNA primer
Label recovered (cpm)
% of total label
Pellet
Supernatant
Pellet
Supernatant
15 603 7 Oli 19 822
4069 424 7411
80 95 72
20 5 28
Biochim. Biophys. Acta, 281 (1972) 216-22. 7
D N A BIOSYNTHESIS IN NUCLEI. I
225
associated with the nucleus r a t h e r t h a n the s u p e r n a t a n t regardless of whether the reaction is primed b y endogenous or exogenous DNA. These experiments c a n n o t dist i n g u i s h whether the exogenous D N A is merely sticking to the nuclear m e m b r a n e or whether the exogenous D N A is a c t u a l l y p e n e t r a t i n g into the nucleus. The products of b o t h exogenously a n d endogenously primed reactions were characterized b y CsC1 d e n s i t y centrifugation. Fig. 4 shows the results of the endogenous reaction a n d indicates t h a t the radioactive peak coincides with the absorbance peak at the b u o y a n t d e n s i t y of rat liver nuclear D N A (p ---- 1.7oo ). This argues s t r o n g l y t h a t the system is synthesizing rat liver nuclear DNA. Fig. 5 shows the results of an e x p e r i m e n t using M. lysodeikticus D N A (p : 1.73I ) as exogenous primer. The d a t a for this e x p e r i m e n t clearly show t h a t the bulk of the DNA, as i n d i c a t e d b y the absorbance, occurs at a b u o y a n t d e n s i t y of rat liver DNA, although only a small a m o u n t of r a d i o a c t i v i t y is recovered in this region. Most of the radioact i v i t y occurs in a peak located at the d e n s i t y of Micrococcus DNA. Thus, b y comparing the d a t a in Fig. 4 a n d 5, it appears t h a t the exogenous primer D N A i n h i b i t s endogenous DNA synthesis while at the same time greatly stimulates synthesis of the exogenous primer. These results lend support to the idea t h a t the a d d i t i o n of D N A to nuclei is comparable to s t u d y i n g a crude solubilized DNA polymerase system.
T
I ,700
I
I
I
I
I
I
1731 j
[
l
[
--016
I
_olo
or2 900
*oo
_ 008
OO4 OO4
O ~
1oo
100 oo2
oo www 40
-w
J
I
I
I
J
4S
50
~5
bO
05
FRACT*ON
o
o
•
0 J
tO
14
le
22
2b
30
34
F'RACTION
Fig. 4- CsC1 centrifugation of the product of the endogenous primed reaction. Assay conditions were as described in the legend to Fig. i except that the assay mixtures were scaled up to 4 ml. E3H]dATP was used in addition to E3H]dTTP and incubation was carried out for 9° min. Each 4-ml tube contained 4.8 mg of nuclear protein. DNA was extracted as described in Experimental and 5°/~g samples run in CsC1 gradients. 0 - 0 , A,60nm; C)--~), cpm. Fig. 5. CsC1 centrifugation of the product of the exogenous primed reaction. Assay conditions were as described in the legend to Fig. I except that the assay mixtures were scaled up to 4 ml, [SH]dATP used in addition to [SHIdTTP, M. lysodeikticus DNA was substituted for calf thymus DNA, and incubation was carried out for 90 min. Each 4-ml tube contained 4.8 mg of nuclear protein. DNA was extracted as described in Experimental and 50 ~ugsamples run in CsC1 gradients. 0 - - 0 , A280 nm; (~--(~, cpm.
Biochim. Biophys. Acta, 281 (1972) 216-227
226
G . s . PROBST Ct a l .
DISCUSSION
A DNA biosynthetic system consisting of isolated rat liver nuclei primed with endogenous DNA has been described. The system can incorporate labeled substrate linearly for at least 2 h. In this period o.o9-o.14 °/o of the input DNA is synthesized. While this value is low in comparison to the capabilities of purified enzymes in vitro, it should be remembered that these nuclei have been isolated from a tissue fairly inactive in DNA synthesis. If we use the data of Naora 46 which gives a mitotic index of 0.0 4 °/o for rats weighing 175 g, use a mitotic time of 0.8 h (ref. 47) and calculate the intermitotic time as described by Lushbaugh *s, we arrive at a figure of 14oo-2ooo h for the in vivo generation time. Since the S phase lasts about 8 h (ref, 49), only 0.4-0.6 % of the nuclei should be engaged in DNA synthesis. During the 2 h incubation we should expect a m a x i m u m of O.lO-O.15 o,~ of the total DNA to be synthesized if, as Lynch et al. lz have suggested, only those nuclei which were synthesizing DNA in vivo at the time of isolation are capable of synthesizing DNA in vitro. Thus, our system appears to be highly efficient and synthesizes DNA at a rate comparable to that in vivo. We hasten to point out that at present we cannot be sure that this synthesis represents DNA replication rather than DNA repair, and work is currently in progress in attempting to distinguish these possibilities. One of our objectives has been to compare DNA synthesis by exogenously and endogenously primed nuclei. The data indicate several minor differences. The most striking difference, however, is the nature of the synthesized product. Our data suggest that most of the synthesis, whether primed b y endogenous or exogenous DNA, is bound to the nucleus. Further, CsC1 density centrifugation indicates that the addition of exogenous DNA suppresses endogenous DNA synthesis. The mechanism by which this occurs is not known. However, it is known that nicking of DNA b y deoxyribonuclease can provide more sites for initiation u,2°'27'44. Since nucleases are present (Table IV), the exogenous DNA template m a y be rapidly activated and act as a much better primer without directly interfering with the endogenous synthesis. The endogenous DNA is in the form of a nucleohistone complex which would make it less subject to nuclease attack. (This in fact is supported by the long term incorporation data (Fig. 2) which showed the endogenously primed product solubilized at a much slower rate than the exogenously primed product). Furthermore, much ol the template m a y not be available for replication. The 37 °C activation phenomenon reported here m a y be related to this. By preincubation there could be a loss of repressors, possibly by protease action, leading to more regions available for synthesis. Alternatively, these bare regions of DNA could now be more susceptible to nuclease action and lead to "activation" of the endogenous DNA. It is also possible that these differences m a y reflect the action of two distinct enzymes. The recent work by Chang and Bollum 4° suggests that there are two DNA polymerases in rabbit nuclei, one tightly bound and the other easily solubilized and identical to the "cytoplasmic" enzyme. We have partially purified the tightly bound enzyme from rat liver 25, and are currently trying to purify it further and determine its role in DNA replication.
Biochim. Biophys. Acta, 281 (i972) 216-227
D N A BIOSYNTHESIS IN NUCLEI. I
227
ACKNOWLEDGEMENTS
We wish to thank Mr David Stalker for his technical assistance, Mr Charles J. Grossman for help in preparing the [3HIDNA, and Miss Diane C. Rein for help with the microscopy. This investigation was supported by Grant NP-24A from the American Cancer Society.
REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49
G. S. Probst, E. B. Koff, S. J. Keller and R. R. Meyer, J. Cell Biol., 47 (197 o) I62a. F. J. Bollum and V. R. Potter, J. Biol. Chem., 233 (1958) 478. R. Mantsavinos and E. S. Canellakis, J. Biol. Chem., 234 (1959) 628. R. Mantsavinos, J. Biol. Chem., 239 (1964) 3431. R. R o y c h o u d h u r y and D. P. Bloch, J. Biol. Chem., 244 (1969) 3359. M. Yoneda a n d F. J. Bollum, J. Biol. Chem., 24o (1965) 3385 • R. M. S. Smillie, H. M. Keir and J. N. Davidson, Biochim. Biophys. Acta, 35 (1959) 389. P. Reichard, Z. N. Canellakis and E. S. Canellakis, J. Biol. Chem., 236 (1961) 2514. F. J. Bollum, J. Biol. Chem., 237 (1962) 1945. R. Greene and D. Korn, J. Biol. Chem., 245 (197 o) 254. R. R o y c h o u d h u r y and D. P. Bloch, J. Biol. Chem., 244 (1969) 3369. W'. E. Lynch, R. F. Brown, T. Umeda, S. G. L a n f r e t h and I. Lieberman, J. Biol. Chem., 245 (197 ° ) 3911. R. W. Kidwell and G. C. Mueller, Biochem. Biophys. Res. Commun., 36 (1969) 756. B. W. Kemper, W. B. P r a t t and L. Aronow, Mol. Pharmacol., 5 (1969) 5o7 . G. W e v e r and S. T. Takats, Biochim. Biophys. Acta, 199 (197 o) 8. R. M. Behki and W. C. Schneider, Biochim. Biophys. Acta, 68 (1963) 34D. Langunoff, Exptl. Cell Res., 55 (1969) 53M. E. Haines, I. R. J o h n s t o n and A. P. Mathias, F E B S Lett., io (197 o) 113. E. N. Brewer and H. P. Rusch, Biochem. Biophys. Res. Commun., 25 (1966) 579. C. C. Richardson, C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem., 239 (1964) 222. T. M. Jovin, P. T. E n g l u n d and A. Kornberg, J. Biol. Chem., 244 (1969) 2996. T. Kornberg and M. L. Gefter, Proc. Natl. Acad. Sci. U.S., 68 (1971) 761. R. E. Moses and C. C. Richardson, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 674. J. Mordoh, Y. Hirota, and F. Jacob, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 773. R. R. Meyer and M. V. Simpson, Proc. Natl. Acad. Sci. U.S., 61 (1968) 13o. R. R. Meyer and M. V. Simpson, Biochem. Biophys. Res. Commun., 34 (1969) 238. R. R. Meyer a n d M. V. Simpson, J. Biol. Chem., 245 (197o) 3426. R. R. Meyer, E. Bikoff, C. C. Teitz and S. Biedenbach, J. Cell Biol., 43 (1969) 92a. P. Parsons a n d M. V. Simpson, Science, 155 (1967) 91M. H. Karol, a n d M. V. Simpson, Science, 12 (1968) 47 o. R. R. Meyer and S. J. Keller, Anal. Biochem., 46 /I972) 332. G. Blobel and V. R. Potter, Science, 154 (1966) 1662. K. S. Kirby, Methods in Enzymology, 12 (1968) 87. R. Anet a n d D. R. Strayer, Biochem. Biophys. Res., Commun., 34 (1969) 328. O. H. Lowry, N . J . Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265. K. Burton, Biochem. J., 62 (1956) 315 . H. M. Keir, Prog. Nucleic Acid Res. Mol. Biol., 4 (1965) 81. J. S. Krakow, C. Coutsogeorgopoulos and E. S. Canellakis, Biochim. Biophys. Acta, 55 (1962) 639. K. I. Kato, J. M. Concalves, G. E. H o u t s and F. J. Bollum, J. Biol. Chem., 242 (1967) 2780. L. M. S. Chang and F. J. Bollum, J. Biol. Chem., 246 (1971) 9o9. M. E. G o t t e s m a n and E. S. Canellakis, J. Biol. Chem., 241 (1966) 4339. F. J. Bollum, E. Groeninger and M. Yoneda, Proe. Natl. Acad. Sci. U.S., 51 (1964) 853. L. M. S. Chang, Biochem. Biophys. Res. Commun., 44 (1971) 124. T. Okazaki a n d A. Kornberg, J. Biol. Chem., 239 (1964) 259. M. Furlan, M. Jericijo and A. Suchar, Biochim. Biophys. Acta, 167 (1968)154. H. Naora, J. Biophys. Biochem. Cytol., 3 (1957) 949. A. M. Brues and B. B. Marble, J. Exp. Med., 65 (1937) 15. C. C. L u s h b a u g h , J. Histochem. Cytochem., 4 (1956) 504 • \V. B. Looney, Proc. Natl. Acad. Sci. U.S., 46 (196o) 69o.
Biochim. Biophys. Acta, 281 (1972) 216-227
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97380
DNA BIOSYNTHESIS IN NUCLEI I. CHARACTERIZATION OF DNA SYNTHESIS BY ISOLATED RAT L I V E R NUCLEI USING ENDOGENOUS DNA AS P R I M E R ~ G R E G O R Y S. P R O B S T , E L I Z A B E T H B I K O F F * * , S T E P H E N J. K E L L E R AND R A L P H R. MEYER
Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 4522r (U.S. 4.) (Received May 8th, 1972)
SUMMARY
I. A DNA biosynthetic system consisting of isolated nuclei using endogenous DNA as primer has been characterized. The nuclei are capable of incorporating E3HI d T T P into DNA linearly for 2 h, and incorporation is proportional to protein concentration up to 2 mg/ml. 2. Preincubation at 37 °C greatly enhances incorporation, but heating for 4 min at 65 °C destroys activity. The pH optimum is 8.0 with Tris-HC1 and 9.0 with glycine-NaOH buffer. The system requires Mg2÷ and is stimulated by ATP, KC1, sucrose and glycerol; but deoxyribonuclease, polyamines, Na~P207, actinomycin D, ethidium bromide and acriflavin inhibit incorporation. 3. Addition of native DNA to the reaction stimulates incorporation 5-told. DNA synthesis by endogenously and exogenously primed nuclei has been compared. CsCI density gradient centrifugation indicates that endogenously primed reactions synthesize rat liver DNA, but addition of exogenous DNA causes inhibition of endogenous DNA synthesis in favor of the exogenous DNA.
INTRODUCTION
In recent years considerable interest has been focused on the mechanism of DNA replication in the eukaryotic chromosome. While an ever-increasing number of studies of crude and purified DNA polymerase have appeared z-n, there have been surprisingly few studies of DNA synthesis by isolated nuclei. The use of intact nuclei offers the advantage of an in vitro system which can be manipulated while at the same time provides a native state at the site of replication which closely approximates that in vivo. While a few reports using nuclear systems have been published 1~-19, in most cases exogenous DNA has been added. Thus, it can be questioned whether the Abbreviation: BBOT, 2,5-bis(2-(tert-butylbenzoxaxolyl)) thiophene. * A p r e l i m i n a r y r e p o r t of p a r t of this w o r k h a s been published 1. The D N A required for enzymatic activity is referred to as the " p r i m e r " in a general sense since its chemical relationship to the p r o d u c t has not been established. ** P r e s e n t address: D e p a r t m e n t of Biological Sciences, Columbia University, New York, N.Y., lOO2 7.
Biochim. Biophys. Acta, 281 (1972) 216-227
DNA BIOSYNTHESISIN NUCLEI. I
217
characteristics of these systems accurately reflect those of the DNA polymerase in its native state, or those of a crude enzyme extract consisting of solubilized DNA polymerase acting on the exogenous DNA. Furthermore, recent studies have indicated that the DNA polymerase of Escheriehia coli isolated by Kornberg ~° and long considered the DNA replicating enzyme, may actually function in DNA repair 2J, and that there are at least two other DNA polymerases present in E. coli ~ presumably one of which is involved in DNA replication. This suggests an advantage of using whole organelles to synthesize DNA: under these conditions one is more likely (although it is not absolutely certain) to be dealing with the DNA replicating system rather than a DNA repair system. This rationale has also led to the use of toluenized cells to study DNA replication in E. coli 38,2, As part of a continuing study of DNA replication in eukaryotes 25-2~, the present study was undertaken (a) to characterize a DNA biosynthetic system from intact nuclei which uses endogenous DNA as a primer, (b) to compare DNA synthesis in nuclei primed by endogenous and exogenous DNA, (c) to serve as a basis for comparison to DNA synthesis by purified nuclear DNA polymerase, and (d) to serve as a basis for comparison" to DNA synthesis by isolated mitochondria 29,3°.
EXPERIMENTAL PROCEDURE
Matermls Unlabeled deoxyribonucleoside triphosphates, ATP, p-nitrophenyl phosphate, and neutral acriflavin were purchased from Sigma Chemical Co., St. Louis, Mo. Thymidine-5'-monophospho-p-nitrophenyl ester, thymidine-3'-monophospho-p-nitrophenyl ester, spermine, spermidine, pronase A and ethidium bromide were obtained from Calbiochem, Los Angeles, Calif. Actinomycin D and bovine serum albumin were purchased from Mann Research Labs, New York, N. Y., while calf thymus DNA, ribonucleases A and T 1 and deoxyribonuclease were obtained from Worthington Biochemical Corp., Freehold, N. J. CsC1 (optical grade) was a product of Gallard and Schlesinger Chemical Co., Carle Place, N. Y. and Micrococcus lysodeikticus and Clostridium per]ringens DNA was a product of Miles Laboratories, Elkhart, Ind. I3HIdTTP, E3H~dATP, I~HIdGTP, and [*HJthymine were obtained from New England Nuclear Corp., Boston, Mass., and E3HIdATP and [SHIdCTP from Schwarz BioResearch, Orangeburg, N. Y. Radioisotopes supplied in 5 ° % ethanol were used directly without removal of the ethanol. Radioactive DNA was prepared from E. coli strain 15 T - H - U - using ~SHJthymine, and activated DNA was prepared as described previously 2~. Denatured DNA was prepared by heating samples at IOO °C for 15 rain followed by rapid cooling. Methods Enzyme assays. DNA polymerase assays were carried out in 12 mm × 75 mm glass tubes in a final reaction volume of 125/A. Assays components are given in the legends. After incubation, IOO/A samples were spotted on filter paper discs, precipitated in io % trichloroacetic acid, and processed for liquid scintillation counting in " Details of these e x p e r i m e n t s will be published in a later c o m m u n i c a t i o n . A preliminary r e p o r t has been published 28.
Biochim. Biophys. Acta, 281 (1972) 216-227
218
G.S. PROBST et al.
2,5-bis-(2-(tert-butylbenzoxaxolyl)) thiophene (BBOT)-toluene as described previously 31. Assays for acid phosphatase, alkaline phosphatase, acid phosphodiesterase I, alkaline phosphodiesterase I, phosphodiesterase II, deoxyribonuclease I and II were performed as before 2~. Isolation of nuclei. In order to obtain large quantities of nuclei, the Blobel and Potter 32 method was scaled up and modified. In a typical preparation 9° g of liver from rats weighing 175-200 g were homogenized with a Potter-Elvehjem homogenizer in a final volume of 270 ml of 0.25 M sucrose in Buffer A (0.05 M Tris-HC1 buffer (pH 8.o), 0.025 M KC1, 0.005 M 2-mercaptoethanol, o.ooi M EDTA, and 0.005 M magnesiumacetate). In some experiments 0.005 M CaC12 was substituted for magnesiumacetate ("calcium nuclei"). The homogenate was passed through four layers of cheesecloth and centrifuged at 800 x g for IO rain. The nuclear pellets were washed twice and suspended in 45 ml of 0.25 M sucrose Buffer A to which was then added 9 ° ml of a 2. 3 M sucrose solution made up in Buffer A. After thorough mixing, 45 ml of this suspension was overlaid onto a 15 ml cushion of 2. 3 M sucrose in Buffer A in each of the tubes. Centrifugation was carried out in a Spinco SW-25.2 rotor at 25 ooo rev./min for 2 h. The pellets were taken up in 20 ml of Buffer A containing 20 *I,, glycerol and frozen in liquid N 2 or at 20 °C. The yield ~f ruclei by this method was about 7 ° °'o as estimated from the recovery of DNA from the filtered homogenate. CsC1 centrifugation. A standard reaction mixture was scaled up to 4.o ml, IZHIdATP was used as a label in addition to [~HIdTTP, and incubation was carried out for 9 ° rain. The nuclei were lysed in 1. 7 % sodium dodecyl sulfate and the DNA extracted by a modified method of Kirby z3. The crude DNA was spooled from ethanol and dried over N 2. The DNA was dissolved in 0.03 M NaC1 0.o03 M sodium citrate, treated with 20/~g/ml ribonuclease A and 50 units/ml of ribonuclease T 1 for 3° rain at 37 °C, re-extracted with phenol, spooled from ethanol, dried over N 2 and dissolved again in 0.03 M NaCl-o.oo 3 M sodium citrate. Samples of 5 °/~g of DNA in CsC1 were run in the Spinco SW 5oL rotor for 60 h, using a "relaxed gradient TM. Five drop fractions were collected, diluted to I ml and the absorbance at 260 nm determined. Carrier DNA (75/~g) was added to the samples which were then spotted onto filter paper discs and prepared for liquid scintillation counting. Densities were determined with an Abbe refractometer. Other procedures. Protein concentrations were measured by the Lowry reaction using bovine serum albumin as a standard and DNA by the diphenylamine procedure ~6 using calf thymus DNA as a standard.
RESULTS
Nuclear purity and stability The purity of the preparations was checked by phase contrast and election microscopy. The modified Blobel and Potter method routinely yielded large quantities of nuclei free of red blood cells, whole liver cells and mitochondria. The nuclei as isolated are very stable when stored in liquid N 2 in buffers containing 20 O//oglycerol or sucrose. Nuclei incubated at 37 °C from 0.5-6 h prior to assay showed a marked increase Biochim. Biophys. Acta, 281 (1972) 216-227
DNA
219
BIOSYNTHESIS IN NUCLEI. I
in the ability to incorporate [3H]dTTP (Fig. I). Maximal stimulation (4.5-fold) was noted for the endogenous system with preincubation for 2 h at 37 °C. Similar results were found with the exogenously primed system, showing a 1.4-fold maximal increase in incorporation after 1-2 h of preincubation, and essentially no loss of maximal activity up to 6 h of preincubation (Fig. I). In contrast, 4 min incubation at 55 °C resulted in 5 ° % loss of activity and at 60 °C all activity was lost. I
I
I
I
I
I 3o
400
I
I
I
2s
300
o oc
c= 20C
,..
15
d
10C
1
I
I
I
I
J
I
0
1
2
3
4
5
6
HOURS
I
6O
I
120
I
1110
240
MINUTE~
Fig. I. E f f e c t of p r e i n c u b a t i o n of nuclei on n u c l e a r D N A s y n t h e s i s . Nuclei s t o c k w a s i n c u b a t e d a t 37 °C for t h e t i m e s i n d i c a t e d in t h e figure. S a m p l e s of 25/~1 were w i t h d r a w n a n d a s s a y e d for D N A p o l y m e r a s e a c t i v i t y for i h. T h e s t a n d a r d r e a c t i o n m i x c o n t a i n e d t h e following c o m p o n e n t s in a final v o l u m e of 125 ~1:o.o25 M Tris-HC1 b u f f e r (pH 8.o), 0.0o 5 M 2 - m e r c a p t o e t h a n o l , o.oo 5 M E D T A , 0.025 M KC], o.oi M m a g n e s i u m acetate, o.oo2 M A T P , o.o15 m M each of d A T P , d C T P , d G T P , a n d [ 3 H ] d T T P (specific a c t i v i t y , 1.41 Ci/mmole), a n d 15 % glycerol. W h e n e x o g e n o u s p r i m e r w a s used, n a t i v e calf t h y m u s D N A w a s a d d e d a t IOO ~ug/ml. O - O , w i t h e x o g e n o u s p r i m e r ; 0-0, with endogenous primer; ~, represents O and • simultaneously. Each assay contained i II lug of n u c l e a r protein. Fig. 2. I n c o r p o r a t i o n of E3H]dTTP into D N A b y isolated nucei over a 4-h period. T h e s t a n d a r d r e a c t i o n m i x t u r e as d e s c r i b e d in t h e legend to Fig. i w a s scaled u p to 4 m], a n d triplicate ioo/~1 s a m p l e s were w i t h d r a w n a t t h e t i m e s i n d i c a t e d in t h e figure. T h e r e a c t i o n m i x t u r e c o n t a i n e d 984 l, gllnl of n u c l e a r protein. O O , w i t h e x o g e n o u s p r i m e r ; 0 - • , with endogenous primer.
E//ect o/concentration o/nuclei Incorporation of labeled substrate into DNA was proportional to the amount of nuclei added up to approx. 2 mg/ml of protein (400/,g/assay).
Time course o/incorporation Incorporation of [3H]dTTP into DNA was fairly linear for a period of at least 2 h in nuclei primed with endogenous DNA and 1.5 h with exogenous DNA (Fig. 2). J Longer incubation led to a loss of labeled product. One of the malor advantages of our system is the ability to incorporate labeled substrate for long periods of time (2 h). Others using endogenously primed nuclear systems have obtained linear Biochim. Biophys. Acta, 281 (1972) 216-227
G . S . PROBST et al.
220
incorporation for only 5-4 o min 12'14'1s or have obtained very low levels of incorporation. We obtained I6~ pmoles/mg protein compared with 2.2-52.8 pmoles/mg of protein reported by others under normal conditions. We can obtain 8o5 pmoles/mg of protein with preincubated nuclei.
E//ect o~ exogenous DNA Addition of calf thymus DNA stimulates the reaction several fold which is consistent with previous studies 16-19. The degree of stimulation with DNA from several sources shows considerable variation (Table I). Native DNA was more effective than denatured DNA (Table II). Enzyme saturation is reached at a concentration of TABLE I ACTIVITY OF RAT L1VER NUCLEI USING VARIOUS D N A PRIMERS R e a c t i o n m i x t u r e s c o n t a i n e d t h e s t a n d a r d a s s a y c o m p o n e n t s as d e s c r i b e d in t h e l e g e n d to Fig. for e x o g e n o u s l y p r i m e d r e a c t i o n s e x c e p t t h a t each t u b e c o n t a i n e d t h e t y p e of D N A as g i v e n in t h e t a b l e , a n d i n c u b a t i o n w a s c a r r i e d o u t for I h. E a c h t u b e c o n t a i n e d 57.6/~g of n u c l e a r prot e i n. " A c t i v a t e d " D N A was p r e p a r e d b y l i m i t e d d e o x y r i b o n u c l e a s e digestion~L
Type of DNA
Biological source (/ DNA
Incorporation of [~H]dT TP pmoles %
Mammalian
Calf t h y m u s " A c t i v a t e d " calf t h y m u s R a t l i v e r nuclei Novikoff hepatoma
i o.o 19.2 6.83 9.82
too 192 68. 3 98.2
Algal
Euglena gracilis Chlamydomonas reinhardii E. coli M. lysodeikticus C. perfringens
3.89 2.81
38.9 28.1
6.78 5.78 8.78
67.8 57.8 87.8
Bacterial
TABLE
II
DNA SYNTHESIS The c o m p l e t e a s s a y m i x t u r e w a s as g i v e n in t h e l e g e n d t o Fig. i, e x c e p t t h a t t h e i n c u b a t i o n t i m e was I h. O m i s s i o n s or a d d i t i o n s t o t h i s were as g i v e n in t h e Table. E a c h a s s a y t u b e c o n t a i n e d 115 to 12o/~g of n u c l e a r p r o t e i n . F o r t h e e n d o g e n o u s p r i m e d r e a c t i o n i o o % a c t i v i t y w a s equiv a l e n t to 1.5-5.I p m o l e s a n d for t h e e x o g e n o u s p r i m e d r e a c t i o n 8.6-13.2 pmoles. SOME REQUIREMENTS FOR
System
Complete -- A T P KC1 - - M g ~+ -- g l y c e r o l --dATP -- d C T P --dGTP - - d A T P , dCTP, d G T P mercaptoethanol --EDTA +spermine (25o/~M) (375/~M) + s p e r m i d i n e (250/tM) (375 #M) --native DNA+denatured -
-
-
-
DNA
Activity (%) Endogenous primer
Exogenous primer
IOO 8 79 5 13 82 7o 47 38 99 98 9 5 4° 32 --
I oo 5 84 ~a 21 76 83 55 55 99 99 4° lo 79 79 37
Biochim. Biophys. Acta, 28I (1972) 216-227
DNA
BIOSYNTHESIS IN NUCLEI. I
221
about 2oo/~g/ml using native DNA but only 75/,g/ml for denatured DNA (Table II). Further increases in DNA concentration begin to inhibit DNA synthesis.
pH e//ects The effects of pH over the range 6.o-11.o using several different buffers were studied. With endogenously primed nuclei, Tris-HC1 or glycine-NaOH buffers were equally effective, although the pH optimum was considerably different (pH 8.0 and 9.0, respectively). With exogenously primed nuclei, the pH optima were approximately the same as with endogenously primed nuclei; however, the activity was almost 2-fold greater with Tris-HC1 than with glycine-NaOH buffer.
E/[ects o/divalent cations Mg~+ is required for DNA synthesis and has an optimum at 5-15 mM (Table II). An absolute requirement could not be demonstrated in this experiment because the nuclei are suspended in a MgS+-containing buffer. An absolute requirement could be demonstrated using Ca 2+ nuclei. Mn 2+ was only lO-29 % effective as Mg2+ at an optimum of o.5-1. 5 raM, and Ca 2+ was ineffective. However, Ca2+-isolated nuclei were consistently more active than Mg2+-isolated nuclei and produced different Mg2+ optima for endogenously and exogenously primed reactions.
E//ect o~ glycerol and sucrose
(~
Glycerol and sucrose stimulated incorporation of labeled substrate into DNA (Fig. 3). For the endogenous system, a 6-fold stimulation was obtained with 25 %
20
i
I
I
I
I
I
I
I
I
I
I
I
is
Z 0
o
t
I
I
I
I
1
5
lo
15
2o
25
3o
MINUTES
Fig. 3- The effects of glycerol and sucrose on D N A synthesis b y isolated nuclei. N u c l e i w e r e washed 3 times in Buffer A prior to use. The a s s a y m i x t u r e w a s t h e same as described in the legend to Fig. I except i n c u b a t i o n was for i h a n d glycerol was excluded. E i t h e r glycerol or sucrose w a s added to t h e individual reaction t u b e s to o b t a i n the c o n c e n t r a t i o n s indicated in the figure, a n d each reaction contained 12o p g of nuclear protein. /x_/x, sucrose w i t h exogenous p r i m e r ; A - A , sucrose w i t h endogenous primer; 0 - 0 , glycerol w i t h exogenous primer; Q - O , glycerol w i t h endogenous primer.
Biochim. Biophys. Acta, 281 (1972) 216-227
222
G . S . PROBST et al.
glycerol, and 7-fold with 3 ° % sucrose. Due to pipetting difficulties, concentrations above 3 ° o/jo could not be tested; thus, no optimum was found for the endogenous system with sucrose. With exogenous primer, glycerol gave a 4-fold stimulation at 15 %, and sucrose gave a 7-fold increase at 2 5 %. The role of sucrose or glycerol in promoting this stimulation is unknown. Whether it acts as a stabilizer of the enzymes involved in replication or if it plays a more central role remains to be determined.
E]/ect o/KCl Monovalent cations have been shown to be moderately stimulatory with most mammalian DNA polymerases studied ~v and highly stimulatory in the case of rat liver 25'27 and calf liver mitochondrial DNA polymerases (R. R. Meyer, unpublished data). Using rat liver nuclei, KC1 was found to stimulate to the extent of up to 2-fold at concentrations of 0.025-0.050 M (Table II). This stimulation is largely an ionic strength effect since other salts could be substituted for KC1. Some specific ion effect was observed since NH4C1 was considerably less effective than KC1 or NaC1.
Deoxyribonucleoside triphosphate requirement The effect of excluding one or more deoxyribonucleoside triphosphates is shown in Table II. Considerable activity is obtained which m a y be due to the presence of a deoxyribonucleoside triphosphate pool or the formation of small amounts of them by nucleoside kinases 14. There m a y be also limited addition of the labeled nucleoside triphosphates to the ends of DNA chains or there m a y be a terminal addition enzyme. present 3s-42. If a terminal addition enzyme is present, it can be questioned to what extent the incorporation in the presence of all four deoxyribonucleoside triphosphates represents DNA polymerase activity and to what extent it represents terminal addition. We feel we are measuring DNA polymerase for the most part, since terminal addition enzymes are primarily poly(A) polymerases 42, and there is recent evidence that terminal addition enzyme m a y be unique to calf thymus 43. Also, deoxyribonucleoside triphosphates inhibit the purified calf terminal addition enzymes which have been studied nv.41. Incorporation can be inhibited 80 % with actinomycin D (see below), and this antibiotic does not inhibit terminal addition enzymes '~.
A TP requirement Isolated nuclei are stimulated by ATP (Table II), with an optimum at 2- 3 mM. Similar effects of ATP have been noted for other nuclear systems 15'1v as well as partially purified DNA polymerases ~5,27. The role of ATP probably lies in inhibiting deoxyribonucleoside triphosphatases or effecting regeneration ot the triphosphate. No purified DNA polymerase has been found to require ATP, and in the case of rat liver mitochondrial DNA polymerase, ATP was no longer required after removal of a deoxyguanosine triphosphatase during purification 27.
E]/ect o/polyamines In the isolated nuclei of Physarum polycephalumxg, spermine greatly stimulates DNA synthesis. In the rat liver nuclei system, polyamines inhibit DNA synthesis (Table II). Brewer and Rusch 19 suggest that spermine promotes penetration of the exogenous primer into the nucleus or activates latent polymerase in the nucleus. Our results suggest that this effect m a y be species specific. Biochim. Biophys. Acta, 281 (1972) 216-227
DNA
BIOSYNTHESIS IN NUCLEI. I
223
Effect o/inhibitors The isolated nuclear system is sentitive to several known inhibitors of DNA polymerase as shown in Table I I I . Addition of sodium pyrophosphate, deoxyribonuclease I, actinomycin D, ethidium bromide or acriflavin markedly decrease activity, although ribonuclease and pronase have virtually no effect. The results of several treatments on the product of the reaction is also given. Treatment of the acid-precipitable product with hot trichloroacetic acid or deoxyribonuclease leads to almost complete solubilization of the product. Ribonuclease and pronase are ineffective. These studies serve to identify the product of the reaction as DNA. TABLE III EFFECTS OF ADDITION OF VARIOUS SUBSTANCES ON D N A SYNTHESIS BY RAT LIVER NUCLEI T h e c o n t r o l t u b e s c o n t a i n e d t h e s t a n d a r d a s s a y c o m p o n e n t s as d e s c r i b e d in t h e l e g e n d t o Fig. I e x c e p t t h a t t h e c o n c e n t r a t i o n of p r o t e i n w a s 3 o 0 / , g / t u b e , a d d i t i o n s were m a d e as g i v e n in t h e t a b l e a n d i n c u b a t i o n w a s for I h. I n E x p t B, a f t e r i n c u b a t i o n , t h e t u b e s w e re h e a t e d to 65 °C for 4 rain a n d cooled to i n a c t i v a t e t h e e n z y m e s . A d d i t i o n s as l i s t e d in t h e t a b l e w e re m a d e a n d i n c u b a t i o n c o n t i n u e d for i h a t 37 °C e x c e p t for t h e t r i c h l o r o a c e t i c a c i d t u b e s w h i c h were h e a t e d a t 90 °C for i o min.
Experiment
(A) a d d i t i o n s before i n c u b a t i o n Control I +deoxyribonuclease (80/tg/ml) + r i b o n u c l e a s e (80 p g / m l ) + N a ~ P z O 7 (4 ° mM) + e t h i d i u m b r o m i d e (500/~M) + a c r i f l a v i n (500/*M) + a c t i n o m y c i n D ~5oo/iM) (B) A d d i t i o n s a f t e r i n c u b a t i o n Control II + d e o x y r i b o n u c l e a s e (8o/~g/ml) + r i b o n u c l e a s e (80/~g/ml) + p r o n a s e (80/~g/ml) + t r i c h l o r o a c e t i c a c i d ( i o %)
Endogenous primer
Exogenous primer
[aH]dTTP incorporated (pmoles )
% control activity
[3H]dTTP incorporated (pmoles )
% control activity
IOO,O
21.o
IOO.O
o 8.5 0.03 o-76 2-44 2.17
8. 9
o 95.5 0. 3 8.5 27.4 24.3
o.15 21.9 0.06 5-7 lO-3 6.5
0. 7 lO4.2 0.2 27.1 49.0 3o.9
9.8
IOO.O
23. 9
IOO.O
o.oi
9.6 i3. 3 0.22
O.I
97.9 135. 7 2.2
0.25
24.0 24.2 o.o 7
I.O
lOO.4 i o i .2 0.2
Additional enzyme activities in nuclei Since phosphatases and nucleases have been shown to affect DNA synthesis n,20,27,~, nuclei were tested for the presence of these enzymes. The results, summarized in Table IV, indicate that nuclei contain phosphatases, phosphodiesterases and nucleases. In addition to these enzymes, nucleoside kinase, and nucleoside diphosphate kinase have been detected b y others 14, as well as a neutral protease in calf thymus nuclei 45. Thus, the role of these enzymes must be taken into consideration with interpreting the results of E3HJdTTP incorporation experiments.
Nature o[ the synthesized product Since the addition of native DNA can increase incorporation over 5-fold, the question arises as to the mechanism of this stimulation. There are several possibilities including (a) the exogenous DNA stimulates endogenous DNA synthesis, (b) exoBiochim. Biophys. Acta, 281 (1972) 216-227
224
G.S. PROBST et al.
TABLE IV ADDITIONAL
ENZYME
ACTIVITIES
IN ISOLATED
RAT LIVER
NUCLEI
Pbosphomonoesterase, phosphodiesterase and deoxyribonuclease activities were determined as described previously2s. Each assay tube contained 222/tg of nuclear protein. For the esterase i unit of activity is equivalent to the release of i pnlole of p-nitrophenol/h at 37 °C. For deoxyribonuclease I unit of activity is equivalent to the release of i nmole of nucleotide per 3° min at 37 °C. Enzyme
Activity (units/rag protein)
Alkaline phosphatase Acid phosphatase Alkaline phosphodieterase 1 Acid phosphodiesterase I Phosphodiesterase II
0.044 0.293 o.o73 0.038 o.oi9
Acid deoxyribonuclease native DNA substrate denatured DNA substrate Alkaline deoxyribonuclease native DNA substrate denatured DNA substrate
14.8 I33.o 12. 4 I O I .O
genous D N A is being s y n t h e s i z e d outside of t he nucleus b y solubilized D N A polymerase, or (c) t h e exogenous D N A enters th e nucleus and is synthesized b y t h e endogenous D N A polymerase. To test these possibilities, reactions were scaled up, an d after i n c u b a t i o n , th e d i s t r i b u t i o n of r a d i o a c t i v e label b et w een the s u p e r n a t a n t and t h e pellet was d e t e r m i n e d . In a n o t h e r set of tubes, E3HIDNA was a d d e d as p r i m e r a nd I 3 H I d T T P was replaced b y u n l a b e l e d d T T P . These tubes were i n c u b a t e d an d t h e n s e p a r a t e d into s u p e r n a t a n t a n d pellet fractions. Th e results of these e x p e r i m e n t s are shown in Tab l e V. T h e d a t a suggest t h a t w i t h endogenous primer almost all of the synthesis occurs in t h e nucleus. T h e 5 % of r a d i o a c t i v i t y found in t h e s u p e r n a t a n t m a y be due to f r a g m e n t e d nuclei. W i t h e x o g e no u sl y p r i m e d reaction, only 20 o//o of t h e label was f o u n d outside of t h e nucleus, p r e s u m a b l y due to D N A polymerase w hich h a d leached out. T h e bulk of t h e label, however, is associated w i t h t h e nucleus. T h e results of the [ 3 H I D N A p r i m e d e x p e r i m e n t s clearly show t h a t g r e a t e r t h a n 7 ° % of the p r i m e r D N A binds to th e nucleus, i n d i c a t i n g t h a t the bulk of the synthesis is
TABLE V DISTRIBUTIONOF RADIOACTIVEDNA BETWEEN SUPERNATANTAND NUCLEARPELLET Exogenous and endogenous reactions (A and B) were run as described in the legend to Fig. i except that they were scaled up to i ml, 1.14 mg of nuclear protein was used and incubation time was I h. At the end of the incubation the tubes were centrifuged at 800 × g to pellet the nuclei, and the distribution of radioactivity between the supernatant and pellet determined. In a third series of tubes (C) [SHIDNA was added, [aH]dTTP omitted and the distribution of label determined after incubation Experimental condition
(A) Exogenous primer (B) Endogenous primer (C) ?H]DNA primer
Label recovered (cpm)
% of total label
Pellet
Supernatant
Pellet
Supernatant
15 603 7 Oli 19 822
4069 424 7411
80 95 72
20 5 28
Biochim. Biophys. Acta, 281 (1972) 216-22. 7
D N A BIOSYNTHESIS IN NUCLEI. I
225
associated with the nucleus r a t h e r t h a n the s u p e r n a t a n t regardless of whether the reaction is primed b y endogenous or exogenous DNA. These experiments c a n n o t dist i n g u i s h whether the exogenous D N A is merely sticking to the nuclear m e m b r a n e or whether the exogenous D N A is a c t u a l l y p e n e t r a t i n g into the nucleus. The products of b o t h exogenously a n d endogenously primed reactions were characterized b y CsC1 d e n s i t y centrifugation. Fig. 4 shows the results of the endogenous reaction a n d indicates t h a t the radioactive peak coincides with the absorbance peak at the b u o y a n t d e n s i t y of rat liver nuclear D N A (p ---- 1.7oo ). This argues s t r o n g l y t h a t the system is synthesizing rat liver nuclear DNA. Fig. 5 shows the results of an e x p e r i m e n t using M. lysodeikticus D N A (p : 1.73I ) as exogenous primer. The d a t a for this e x p e r i m e n t clearly show t h a t the bulk of the DNA, as i n d i c a t e d b y the absorbance, occurs at a b u o y a n t d e n s i t y of rat liver DNA, although only a small a m o u n t of r a d i o a c t i v i t y is recovered in this region. Most of the radioact i v i t y occurs in a peak located at the d e n s i t y of Micrococcus DNA. Thus, b y comparing the d a t a in Fig. 4 a n d 5, it appears t h a t the exogenous primer D N A i n h i b i t s endogenous DNA synthesis while at the same time greatly stimulates synthesis of the exogenous primer. These results lend support to the idea t h a t the a d d i t i o n of D N A to nuclei is comparable to s t u d y i n g a crude solubilized DNA polymerase system.
T
I ,700
I
I
I
I
I
I
1731 j
[
l
[
--016
I
_olo
or2 900
*oo
_ 008
OO4 OO4
O ~
1oo
100 oo2
oo www 40
-w
J
I
I
I
J
4S
50
~5
bO
05
FRACT*ON
o
o
•
0 J
tO
14
le
22
2b
30
34
F'RACTION
Fig. 4- CsC1 centrifugation of the product of the endogenous primed reaction. Assay conditions were as described in the legend to Fig. i except that the assay mixtures were scaled up to 4 ml. E3H]dATP was used in addition to E3H]dTTP and incubation was carried out for 9° min. Each 4-ml tube contained 4.8 mg of nuclear protein. DNA was extracted as described in Experimental and 5°/~g samples run in CsC1 gradients. 0 - 0 , A,60nm; C)--~), cpm. Fig. 5. CsC1 centrifugation of the product of the exogenous primed reaction. Assay conditions were as described in the legend to Fig. I except that the assay mixtures were scaled up to 4 ml, [SH]dATP used in addition to [SHIdTTP, M. lysodeikticus DNA was substituted for calf thymus DNA, and incubation was carried out for 90 min. Each 4-ml tube contained 4.8 mg of nuclear protein. DNA was extracted as described in Experimental and 50 ~ugsamples run in CsC1 gradients. 0 - - 0 , A280 nm; (~--(~, cpm.
Biochim. Biophys. Acta, 281 (1972) 216-227
226
G . s . PROBST Ct a l .
DISCUSSION
A DNA biosynthetic system consisting of isolated rat liver nuclei primed with endogenous DNA has been described. The system can incorporate labeled substrate linearly for at least 2 h. In this period o.o9-o.14 °/o of the input DNA is synthesized. While this value is low in comparison to the capabilities of purified enzymes in vitro, it should be remembered that these nuclei have been isolated from a tissue fairly inactive in DNA synthesis. If we use the data of Naora 46 which gives a mitotic index of 0.0 4 °/o for rats weighing 175 g, use a mitotic time of 0.8 h (ref. 47) and calculate the intermitotic time as described by Lushbaugh *s, we arrive at a figure of 14oo-2ooo h for the in vivo generation time. Since the S phase lasts about 8 h (ref, 49), only 0.4-0.6 % of the nuclei should be engaged in DNA synthesis. During the 2 h incubation we should expect a m a x i m u m of O.lO-O.15 o,~ of the total DNA to be synthesized if, as Lynch et al. lz have suggested, only those nuclei which were synthesizing DNA in vivo at the time of isolation are capable of synthesizing DNA in vitro. Thus, our system appears to be highly efficient and synthesizes DNA at a rate comparable to that in vivo. We hasten to point out that at present we cannot be sure that this synthesis represents DNA replication rather than DNA repair, and work is currently in progress in attempting to distinguish these possibilities. One of our objectives has been to compare DNA synthesis by exogenously and endogenously primed nuclei. The data indicate several minor differences. The most striking difference, however, is the nature of the synthesized product. Our data suggest that most of the synthesis, whether primed b y endogenous or exogenous DNA, is bound to the nucleus. Further, CsC1 density centrifugation indicates that the addition of exogenous DNA suppresses endogenous DNA synthesis. The mechanism by which this occurs is not known. However, it is known that nicking of DNA b y deoxyribonuclease can provide more sites for initiation u,2°'27'44. Since nucleases are present (Table IV), the exogenous DNA template m a y be rapidly activated and act as a much better primer without directly interfering with the endogenous synthesis. The endogenous DNA is in the form of a nucleohistone complex which would make it less subject to nuclease attack. (This in fact is supported by the long term incorporation data (Fig. 2) which showed the endogenously primed product solubilized at a much slower rate than the exogenously primed product). Furthermore, much ol the template m a y not be available for replication. The 37 °C activation phenomenon reported here m a y be related to this. By preincubation there could be a loss of repressors, possibly by protease action, leading to more regions available for synthesis. Alternatively, these bare regions of DNA could now be more susceptible to nuclease action and lead to "activation" of the endogenous DNA. It is also possible that these differences m a y reflect the action of two distinct enzymes. The recent work by Chang and Bollum 4° suggests that there are two DNA polymerases in rabbit nuclei, one tightly bound and the other easily solubilized and identical to the "cytoplasmic" enzyme. We have partially purified the tightly bound enzyme from rat liver 25, and are currently trying to purify it further and determine its role in DNA replication.
Biochim. Biophys. Acta, 281 (i972) 216-227
D N A BIOSYNTHESIS IN NUCLEI. I
227
ACKNOWLEDGEMENTS
We wish to thank Mr David Stalker for his technical assistance, Mr Charles J. Grossman for help in preparing the [3HIDNA, and Miss Diane C. Rein for help with the microscopy. This investigation was supported by Grant NP-24A from the American Cancer Society.
REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49
G. S. Probst, E. B. Koff, S. J. Keller and R. R. Meyer, J. Cell Biol., 47 (197 o) I62a. F. J. Bollum and V. R. Potter, J. Biol. Chem., 233 (1958) 478. R. Mantsavinos and E. S. Canellakis, J. Biol. Chem., 234 (1959) 628. R. Mantsavinos, J. Biol. Chem., 239 (1964) 3431. R. R o y c h o u d h u r y and D. P. Bloch, J. Biol. Chem., 244 (1969) 3359. M. Yoneda a n d F. J. Bollum, J. Biol. Chem., 24o (1965) 3385 • R. M. S. Smillie, H. M. Keir and J. N. Davidson, Biochim. Biophys. Acta, 35 (1959) 389. P. Reichard, Z. N. Canellakis and E. S. Canellakis, J. Biol. Chem., 236 (1961) 2514. F. J. Bollum, J. Biol. Chem., 237 (1962) 1945. R. Greene and D. Korn, J. Biol. Chem., 245 (197 o) 254. R. R o y c h o u d h u r y and D. P. Bloch, J. Biol. Chem., 244 (1969) 3369. W'. E. Lynch, R. F. Brown, T. Umeda, S. G. L a n f r e t h and I. Lieberman, J. Biol. Chem., 245 (197 ° ) 3911. R. W. Kidwell and G. C. Mueller, Biochem. Biophys. Res. Commun., 36 (1969) 756. B. W. Kemper, W. B. P r a t t and L. Aronow, Mol. Pharmacol., 5 (1969) 5o7 . G. W e v e r and S. T. Takats, Biochim. Biophys. Acta, 199 (197 o) 8. R. M. Behki and W. C. Schneider, Biochim. Biophys. Acta, 68 (1963) 34D. Langunoff, Exptl. Cell Res., 55 (1969) 53M. E. Haines, I. R. J o h n s t o n and A. P. Mathias, F E B S Lett., io (197 o) 113. E. N. Brewer and H. P. Rusch, Biochem. Biophys. Res. Commun., 25 (1966) 579. C. C. Richardson, C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Chem., 239 (1964) 222. T. M. Jovin, P. T. E n g l u n d and A. Kornberg, J. Biol. Chem., 244 (1969) 2996. T. Kornberg and M. L. Gefter, Proc. Natl. Acad. Sci. U.S., 68 (1971) 761. R. E. Moses and C. C. Richardson, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 674. J. Mordoh, Y. Hirota, and F. Jacob, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 773. R. R. Meyer and M. V. Simpson, Proc. Natl. Acad. Sci. U.S., 61 (1968) 13o. R. R. Meyer and M. V. Simpson, Biochem. Biophys. Res. Commun., 34 (1969) 238. R. R. Meyer a n d M. V. Simpson, J. Biol. Chem., 245 (197o) 3426. R. R. Meyer, E. Bikoff, C. C. Teitz and S. Biedenbach, J. Cell Biol., 43 (1969) 92a. P. Parsons a n d M. V. Simpson, Science, 155 (1967) 91M. H. Karol, a n d M. V. Simpson, Science, 12 (1968) 47 o. R. R. Meyer and S. J. Keller, Anal. Biochem., 46 /I972) 332. G. Blobel and V. R. Potter, Science, 154 (1966) 1662. K. S. Kirby, Methods in Enzymology, 12 (1968) 87. R. Anet a n d D. R. Strayer, Biochem. Biophys. Res., Commun., 34 (1969) 328. O. H. Lowry, N . J . Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265. K. Burton, Biochem. J., 62 (1956) 315 . H. M. Keir, Prog. Nucleic Acid Res. Mol. Biol., 4 (1965) 81. J. S. Krakow, C. Coutsogeorgopoulos and E. S. Canellakis, Biochim. Biophys. Acta, 55 (1962) 639. K. I. Kato, J. M. Concalves, G. E. H o u t s and F. J. Bollum, J. Biol. Chem., 242 (1967) 2780. L. M. S. Chang and F. J. Bollum, J. Biol. Chem., 246 (1971) 9o9. M. E. G o t t e s m a n and E. S. Canellakis, J. Biol. Chem., 241 (1966) 4339. F. J. Bollum, E. Groeninger and M. Yoneda, Proe. Natl. Acad. Sci. U.S., 51 (1964) 853. L. M. S. Chang, Biochem. Biophys. Res. Commun., 44 (1971) 124. T. Okazaki a n d A. Kornberg, J. Biol. Chem., 239 (1964) 259. M. Furlan, M. Jericijo and A. Suchar, Biochim. Biophys. Acta, 167 (1968)154. H. Naora, J. Biophys. Biochem. Cytol., 3 (1957) 949. A. M. Brues and B. B. Marble, J. Exp. Med., 65 (1937) 15. C. C. L u s h b a u g h , J. Histochem. Cytochem., 4 (1956) 504 • \V. B. Looney, Proc. Natl. Acad. Sci. U.S., 46 (196o) 69o.
Biochim. Biophys. Acta, 281 (1972) 216-227