Nature of the deoxyribonucleic acid made by isolated liver nuclei

28

BIOCHIMICA ET B I O P H Y S I C A ACTA

BBA 97422

N A T U R E OF T H E D E O X Y R I B O N U C L E I C ACID MADE BY ISOLATED L I V E R NUCLEI WILLIAM E. LYNCH, TETSUHIKO UMEDA, MASARU UYEDA .aNDIRVING LIEBERMAN Department of Anatomy and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pa. 152z3 (U.S.A.) (Received June 26th, i972)

SUM MARY

To better study the nature of the process of deoxyribonucleotide incorporation by isolated nuclei from regenerating rat liver, improvements have been made in tile assay mixture. The changes include the addition of a Ca 2+binder, ethylene glycol-bis(2-aminoethyl e t h e r ) - N , N ' - t e t r a a c e t a t e (EGTA), a high molecular weight compound such as dextran, and a greater concentration of buffer. The EGTA and increased buffer depress the hydrolysis of DNA during incubation of tile nuclei at 37 °C. Under the new conditions, the initial rate of DNA synthesis is raised by about 3-fold. Additional evidence is given to show that the formation of DNA in vitro is by ttle elongation of the chains that were growing in vivo. The growing points of both strands of DNA appear to be advanced by the isolated nuclei. The approximate rates of advancement in vivo and in vitro were 14oo and 2oo deoxynucleotides per min per strand, respectively.

INTRODUCTION

Evidence has been given 1 to show that isolated nuclei from regenerating rat liver can elongate the DNA chains that were growing in vivo but the possibility was not excluded that the reaction in vitro is a repair rather than a replicative process. Exactly the same results would be obtained if the DNA newly formed in the liver, but not the bulk of the DNA, were susceptible to nucleases that act during isolation or incubation of the nuclei to produce new points of initiation for deoxynucleotide incorporation. If this were so, just as has been found, only nuclei that already contain growing strands would be active in vitro and the incorporated radioactivity would be in close association with the DNA that had just been made in vivo. Two types of repair processes have been considered: first, new chains grow from the 3'-OH ends of nicks or gaps that are formed in the DNA newly-made in vivo; and second, fragments of the nascent DNA made in the liver are excised and the gaps are repaired. Efforts have now been made to distinguish between repair processes and the advancement of the original replication fork. With the test mixture used previously 1, breaks in the DNA are produced during Abbreviations: EGTA, ethylene glycol-bis-(2-aminoethyl ether)-N,N'-tetraacetate. Biochim. Biophys. Acta, 287 (I972) 28-37

DNA

MADE BY ISOLATED NUCLEI

29

incubation at 37 °C as judged by the drop in viscosity of lysates of the liver nuclei and by the properties of the isolated DNA in gradients of alkaline sucrose. Before some of the experiments could be carried out, it was essential to find conditions under which DNA damage is reduced during incubation of the nuclei at 37 °C, The purpose of this report is to describe the improved assay conditions and to show the additional evidence that the product of the isolated liver nuclei represents an elongation of the DNA strands that were growing in the cell.

MATERIALS AND METHODS

Chemicals Labeled compounds were from New England Nuclear. Pronase was from Calbiochem, micrococcal nuclease was from Worthington, and all other unlabeled materials were obtained from Sigma. Micrococcal nuclease (IOO °C, 5 rain) and Pronase (80 °C, IO nfin (ref. 2)) were heated before use. Treatment o/ rats Male albino rats, obtained locally, were freely given food and water and were used when they weighed about IOO g. Partial hepatectomy refers to the removal of about 7° % of the liver (left lateral and median lobes3). Unless otherwise indicated, all injections were made in the tail vein. Isolation o] nuclei and estimation o] DNA synthesis Nuclei were isolated from regenerating liver (19-21 h after partial hepatectomy) by sedimentation in mixtures of sucrose and CaC12 as previously described 1. DNA synthesis was measured at 37 °C in mixtures (0.5 ml) that contained Tris-HC1 buffer (pH 8.2), 3 mM MgCI~, 2 mM ATP, dGTP, dCTP, dATP (each 0.08 raM), o.o16 mM E3H]TTP (0.5 Ci/mmole), and the suspension of intact liver nuclei (o.I ml in 0.3 M sucrose). 2-Mercaptoethanol and KC1, used previously 1, were omitted since they did not enhance the rate of E~H~TTP incorporation. Ethylene glycol-bis(2-aminoethyl ether)-N,N'-tetraacetate (EGTA) (I raM), dextran (Type IooC, 2 %), and cadaverine (2.5 raM) were added as indicated. The radioactivity in nuclear DNA was measured as beforO and all the data were corrected for the incorporation with control mixtures that lacked the three unlabeled deoxynucleotides. DNA was estimated colorimetrically with diphenylaminO. Viscosity measurements Intact nuclei (o.I ml in o.3 M sucrose) were incubated at 37 °C in the same mixtures (0.5 ml) as were used for the estimation of ESH]TTP incorporation (with dextran and cadaverine) except that ATP and the deoxynucleotides were omitted. At the end of the incubation period, the nuclei were lysed by the addition of o.25 ml of 3 % sodium dodecyl sulfate. Viscosities were measured at 24 °C in a CannonManning semi-micro viscometer (size IOO, Cannon Instrument Co.) and sucrose solutions were used as standards. Sedimentation o/ DNA in alkaline sucrose gradients To prepare DNA for the estimation of sedimentation velocities, deproteiniBiochim. Biophys. Acta, 287 (1972) 28-37

3°

W . E . LYNCH Cl al.

zation was done by a procedure that allowed a complete recovery of the DNA. Nuclei were lysed with o.5 % sodium dodecyl sulfate and the lysate was treated with 2 mg of deoxyribonuclease-free Pronase for 3 h at 6o °C. NaOH (i M) was now added to give a final concentration of o.I M and the entire preparation was layered on a gradient of 5 2o % sucrose containing o.i M NaOH, I mM EDTA, and o. 9 M NaC1. Centrifugation was in a Spinco SW-5o.I rotor for 2-6 h (4° ooo rev./min, 2o °C). Sedimentation coefficients of DNA were calculated from Studier 5 equations with T 7 DNA as a marker. In double isotope counting, the overlap of 3H into the ~4C channel was negligible; the overlap of 14C into the 3H channel was 9 %.

RESULTS

Improved test mixture~ Tile viscosities of lysates of nuclei that had been incubated at 37 °C without EGTA were greatly reduced regardless of the concentration of Tris buffer in the test mixtures (Table I). As the table shows, in the presence of EGTA, however, the drop in viscosity was largely prevented when the concentration of Tris was raised to 0.2 M. Similar results were obtained when 0.2 M morpholinopropane sodium sulfonate was used in place of Tris, when mixtures containing EGTA and o.I M Tris were supplemented with NaCI, KC1, or LiC1 (o.I M), and when the nuclei were isolated with solutions of sucrose and Mg2+ instead of sucrose and Ca 2+. TABLE I OF EGTA AND TRIS ON VISCOSITIES OF LYSATES FROM NUCLEI INCUBATED AT 37 oC I n t a c t nuclei (t6o # g of DNA) were incubated in test m i x t u r e s w i t h E G T A and Tris (pH 8.2) as shown. After incubation, the nuclei were lysed w i t h sodium dodecyl sulfate and the viscosities of the lysates were m e a s u r e d as described in Materials and Methods. EFFECTS

Incubation period (rain)

Tris (M)

Viscosity (centipoise) EGTA

+EGT,4

o

0.05 o.2

~3.8 I3.9

I4.1 I4-3

5

0.05 o.i 0.2

3.2 3.4 4.2

8-3 I1.3 12. 4

3°

o.o5 o.I O.2

2.3 2. 5 3.2

3.3 4.0 lO.3

The effects of EGTA and Tris could also be shown by sedimenting the deproteinized DNAs in alkaline sucrose gradients. Bulk DNA from unincubated nuclei or from nuclei that had been kept for 3o min at 37 °C in mixtures containing EGTA and o.2 M Tris sedimented as a single, almost symmetrical peak with an s value of about 5 o. After incubation in a mixture with o.i M Tris and no EGTA, on the other hand, the peak was broader, had an s value of about 3o, and there was a shoulder of smaller molecules that comprised about half of the total DNA. Biochim. Biophys. Acta, 287 (I972) 28-37

DNA MADE BY ISOLATED NUCLEI

31

The final step in the isolation procedure involved the sedimentation of the liver nuclei in 2.2 M sucrose whereupon the nuclei were suspended in 0. 3 M sucrose. Suspension of the nuclei in the 0.3 M solution caused swelling that could be reduced by the inclusion of 2 % dextran (or 4 % "Ficoll") in the sucrose solution. Thus, the average diameter of the nuclei in 2.2 M sucrose was found to be IO/~m with a range of 7-I4/~m. After transfer to 0.3 M sucrose, the average diameter was increased to 16/zm with a range of lO-29 #m, and with 0.3 M sucrose containing 2 % dextran, the comparable values were 12 and I O - i 8 # m . Assuming that all the nuclei were spherical, the increase in nuclear volume in the 0. 3 M sucrose was more than 4-fold whereas in the sucrose-dextran solution it was less than 2-fold. The effects of EGTA, dextran, and cadaverine on the rates of [SH]TTP incorporation by the isolated nuclei were measured in mixtures that contained o.18 M Tris buffer (pH 8.2) (Fig. i). As can be seen from the figure, EGTA increased only the initial rate; dextran stimulated incorporation throughout the period of incubation although its greatest effect was on the initial rate; and cadaverine affected the rate after the first 5-min period only.

+EGTA, DEXTRAN, CADAVERINE •

J

+ EG"TA, DEXTRAN

/" e//

/

+EGTA ~ .

s

I~

°

;

•

NO A D D I T I O ~

,; MINUTES

2; OF

~

~o

INCUBATION

Fig. i. Effects of EGTA, dextran and cadaverine on the kinetics of [SH]TTP incorporation. Intact nuclei (I2O/,g ot DNA) were incubated in test mixtures (o.i8 M Tris buffer (pH 8.2)) containing EGTA, dextran, and cadaverine, as shown. EaH]TTP incorporation was estimated as described in Materials and Methods.

The same results were obtained when morpholinopropane sodium sulfonate was used instead of Tris and when dextran, Type IooC (average tool. wt, 135 ooo), was replaced with 2 °/o dextran of Type 6oC (average mol. wt, 82 400) or Type 500 (average tool. wt, 500 ooo), or with 4 % Ficoll (average tool. wt, 400 ooo). Negligible incorporation of [~H]TTP occurred with normal liver nuclei in complete mixtures and with regenerating nuclei in control mixtures that lacked the three unlabeled deoxynucleotides; after 5 min of incubation, the incorporated radioactivity was about 5 and i °/o, respectively, of that with regenerating nuclei in the experimental mixture. Biochim. Biophys. Acta, 287 (1972) 28-37

32

w.E.

LYNCH et al.

Rates o] elongation o / D N A in vivo and in vitro and pertinent length o/nascent D N A made in vivo The rates of elongation been

measured

of DNA in a variety

at 0.025- 5 #m/min

(refs. 6 - 1 o )

of cultured with

most

mammalian

cells have

of the values

f a l l i n g in

t h e r a n g e o f 0. 5 1.5 # m ( I 5 O O - 4 5 o o n u c l e o t i d e s p e r m i n p e r s t r a n d ) . T o f i n d t h e a v e r a g e r a t e f o r r a t l i v e r n u c l e i in vivo, a n i m a l s w e r e g i v e n [ 3 H ~ t h y m i d i n e f o r I r a i n and

the DNA

was isolated

and hydrolyzed

in such a way

that

internal

molecules

were released as deoxynucleotides, molecules at the 3'-OH ends of strands, as deoxvnucleosides n. The rate of DNA synthesis, calculated from the ratio of I3HITMP to IaHjthymidine, Table

TABLE RATES

was found

to be 14oo nucleotides

per rain per strand

II also shows the results of measurements

made

(Table

II).

in the same way after

II

OF ELONGATION

OF

DNA

BY LIVER

NUCLEI

in viuo

AND

i~ vitro

In vivo: P a r t i a l l y h e p a t e c t o m i z e d r a t s were g i v e n 2oo/,Ci of E I H ] t h y m i d i n e in t h e m e s e n t e r i c vein a t 2o 11 after t h e operation. L i v e r s a m p l e s were r e m o v e d i m i n l a t e r but, as i n d i c a t e d in t h e t e x t , t h e labeling period was t a k e n to be o.5 min. D N A w a s e x t r a c t e d f r o m isolated nuclei with 2 M NaCI e s s e n t i a l l y as described b y Z a m e n h o P 2. T h e D N A w a s t r e a t e d w i t h P r o n a s e a n d proteolysis w a s s t o p p e d w i t h a m i x t u r e of c h l o r o f o r m a n d i s o a m y l alcohol. H y d r o l y s i s of t h e D N A w a s a t 37 'C, first for 2 h w i t h micrococcal n u c l e a s e (7/~g/ml) a n d t h e n for 3 ° m i n w i t h spleen p h o s p h o d i e s t e r a s e (o.2 unit). T h e p r o d u c t s of h y d r o l y s i s were e x a m i n e d b y c h r o m a t o g r a p h y on c o l u n m s of D o w e x - i (C1) (8 °,o cross-linked). T h y m i d i n e w a s collected w i t h o.oo 3 M HC1 a n d T M P was eluted w i t h o.o 5 M HCI. C o u n t i n g w a s in a solution of t o l u e n e a n d T r i t o n X - i o o a n d all t h e c o u n t s applied to t h e c o l u m n s were recovered in t h e t h y m i d i n e a n d T M P areas. W i t h longlabeled D N A , o.i % of t h e t o t a l r a d i o a c t i v i t y was f o u n d as t h y m i d i n e . T h e s e c o u n t s were considered to be d e r i v e d b y t h e h y d r o l y s i s of i n t e r n a l T M P . For this reason, t h e v a l u e s s h o w n for t h y m i d i n e were corrected b y s u b t r a c t i n g o.I % of t h e T M P c o u n t s f r o m t h e gross t h y n l i d i n e c o u n t s . T h e r e s u l t s for t h y m i d i n e before correction are s h o w n in t h e p a r e n t h e s e s . In vitro: P a r t i a l l y h e p a t e c t o m i z e d r a t s were given 2/~moles of 5 - b r o m o - 2 ' d e o x y u r i d i n e in t h e tail vein a t 20 h after t h e o p e r a t i o n a n d liver s a m p l e s were t a k e n 5 rain later. I s o l a t e d nuclei ( a b o u t ~oo ltg of D N A ) were i n c u b a t e d a t 37 °C for t h e i n d i c a t e d t i m e s in t h e c o m p l e t e t e s t m i x t u r e (o.18 IV[ Tris, p H 8.2, E G T A , d e x t r a n a n d c a d a v e r i n e ) e x c e p t t h a t t h e specific activ i t y of t h e ! 3 H ! T T P was raised to i o Ci/mmole. T h e r e a c t i o n s were s t o p p e d b y Gle a d d i t i o n of s o d i u m dodeeyl s u l f a t e a n d t h e D N A s were purified as above. T h e purified D N A s were s h e a r e d in a Virtis h o m o g e n i z e r , d e n a t u r e d b y h e a t i n g a t i o o ~'C for i o rain, a n d c e n t r i f u g e d in a n equilib r i u m g r a d i e n t of n e u t r a l CsC1. T h e d e n s e s t D N A (50 70 % of t h e t o t a l counts), as j u d g e d from t h e a b s o r b a n c e of t h e b u l k D N A at 260 n m , w a s pooled a n d u s e d for hydrolysis. M e a s u r e m e n t s with t o t a l D N A s a m p l e s g a v e r a t e s t h a t were 20-3 ° o{~ lower t h a n t h o s e with t h e dense D N A . E x a c t l y t h e s a m e results were o b t a i n e d w h e n t h e c o n c e n t r a t i o n of mierocoecal nuclease or spleen p h o s p h o d i e s t e r a s e w a s raised 4-fold a n d w h e n t h e t i m e s of i n c u b a t i o n of t h e D N A s a m p l e s with t h e e n z y m e s were increased 4-fold.

Labeling period (,tin)

T.1IP (cpm)

Thymidine (cpm)

In vivo

0.5 0.5

59ooo 55ooo

78 (I4O) 81 (14o)

In vitro

0.25 0-25 0.5 o. 5 i i

3 800 5 ooo 7 IOO xI ooo 16000 69 ooo

69 97 66 14o 78 35 °

(73) (ioo) (73) (i5 o) (94) (420)

7"3It ) ! thymidine Thymidine ratio

Rate (nucleotides/min)

757 680

I514 136o

56 52 lO9 So 206 i()8

224 ~ 208 218" i6o 2o6" I98"*

* T h e s a m e b a t c h of nuclei w a s used. *" A r a t e of 2.5 n u c l e o t i d e s p e r rain w a s f o u n d w h e n t h e t h r e e u n l a b e l e d d e o x y n u c l e o s i d e t r i p h o s p h a t e s were o m i t t e d from t h e r e a c t i o n m i x t u r e .

Biochim. Biophys. dcta, 287 (1972) 28-37

D N A MADE BY ISOLATED NUCLEI

33

briefly labeling the isolated nuclei with [aH]TTP. If it is correct t h a t the nuclei elongated the s t r a n d s t h a t were growing in vivo, the rate in vitro was 200 nucleotides per rain per strand, more t h a n IO % of the rate in the animal. The value in wvo can only be accepted as a n a p p r o x i m a t i o n of the true rate since the calculations involved the use of two correction factors. The first was needed to take into account the n o n - l i n e a r kinetics of labeling of liver D N A in the animal. A b o u t a o.5-min delay in [nH]thymidine incorporation was found after injection of the rat whereupon the rate was linear. For this reason, a I-rain pulse was t a k e n to be e q u i v a l e n t to a o.5-min labeling period. The second factor was required to correct for the EaH~thymidine t h a t was consistently found in hydrolysates of long-labeled D N A a n d t h a t was considered to come from i n t e r n a l deoxynucleotides. The second factor was also applied to the results with the isolated nuclei but, as can be seen from the table, in this case, the corrections were negligible. The deoxynucleotides incorporated b y the isolated liver nuclei have been shown to be in close association with the DNA strands t h a t were growing in vivo 1. To learn a b o u t the length of the nascent strands in vivo with which the counts in vitro become associated, partially hepatectomized rats were given 5-bromodeoxyuridine for 2.5, 5, or IO m i n a n d liver nuclei were isolated a n d allowed to incorporate [nH]TTP for 5 rain. At the e n d of this time, the DNAs were purified, sheared to a n average length of 5600 nucleotides, a n d denatured. Centrifugation in equilibrium gradients of n e u t r a l CsC1 showed t h a t the 8H-labeled DNAs were dense a n d the curves were indistinguishable from one a n o t h e r in spite of the differences in the times the rats h a d been t r e a t e d with 5-bromodeoxyuridine. The results with the three gradients were the same as t h a t shown in Fig. 2A. Thus, the p e r t i n e n t segment of the newly formed D N A made in vivo c a n n o t be larger t h a n 2 m i n worth of DNA, 2800 nucleotides.

~H

14G

3H

]

140

SM

7M

.

Z~

2.~ 251

m

35

40

45

|

3|

35

41

45

e

FRAOTION NUMBER

Fig. 2. Effect of shear of DNA on the association of deoxynucleotides incorporated in vivo and in vitro. A partially hepatectomized rat was labeled with p*C]thymidine for 2 h followed by 5bromodeoxyuridine for 2.5 min. Isolated liver nuclei (23o/~g of DNA) were allowed to incorporate EnH]TTP for io min in the complete test mixture (o.18 M Tris buffer (pH 8.2), EGTA, dextran and cadaverine). After incubation, the DNA was purified with 2 M NaC1 (ref. 12) and it was then sheared in a Virtis homogenizer (setting of 7o V, 7 min). (A) A portion of the sheared DNA was then additionally broken with sonic oscillation (Bronwill Biosonik 111, 0.5 rain at a maximum setting (36) for the microtip). (B) The sheared preparations were heat-denatured and centrifuged in a Spinco SW-5o rotor for 6o h (37 500 rev./min 25 °C) in an equilibrium gradient of neutral CsC1. Collection of 6-drop fractions (75) was taken from the bottom of the tube and counting was done in a solution of toluene and Triton X-ioo. Density decreases from left to right. O-Q), 3H; 0 - 0 , 14C. Biochim. Biophys. Acta, 287 (1972) 28-37

34

w.E. LYNCH et al,

Nature o] the D N A made in vitro The first repair process that was visualized depends upon the formation of a nick or a gap in the DNA chain that was growing in vivo. Such damage should produce a piece of nascent DNA, less than 2800 nucleotides long, that extends from the break to the original growing point. To look for small pieces of nascent DNA, nuclei (26o/~g of DNA) that had been labeled for 1. 5 rain with [3Hlthymidine in vivo were incubated for 30 rain at 37 °C in the standard mixture (with o.18 M Tris, EGTA, dextran, and cadaverine) used to measure DNA synthesis except that all the deoxynucleotide substrates were unlabeled. After incubation, the nuclei were deproteinized and tile DNA was sedimented in an alkaline sucrose gradient as detailed in Materials and Methods. Although more than 98 °/o of the radioactivity of the nuclei was recovered, no pieces of labeled DNA as small as 13 S (2800 nueleotides) were found. In addition, comparison with DNA front nuclei that had not been kept at 37 °C showed that the size of the radioactive DNA (34 S) had not been reduced during incubation. As a control, a 3H-labeled DNA sample was cosedimented with a portion of highly sheared 14Clabeled DNA. No evidence was found for entanglement of the 14C- and :~H-labeled DNAs. The second repair process would involve the excision and replacement in vitro of the deoxynucleotides that had just been incorporated into DNA in the animal. Such a reaction might be expected to lead to the release of acid-soluble nucleotides from the DNA made in vivo. To test this possibility, a partially hepatectomized rat was given I3H]thymidine for 1. 5 min (incorporation of 14oo nucleotides) and the labeled liver nuclei were then isolated and incubated at 37 °C in a complete but nonradioactive test mixture. After 30 min, I M NaOH was added (final concn 0.5 M) and the mixture was then acidified with HC1Q. Only I (~'oof the radioactivity was found to be acid-soluble. Since it could be calculated* that the isolated nuclei had added 18oo deoxynucleotides to each growing strand, the proposed repair mechanisna could be ruled out, requiring as it would that all the radioactive DNA be converted to an acid-soluble form. An additional argument against the second type of repair process came from studies of the effects of shear on DNA labeled in vivo and in vitro. A partially hepatectomized rat was given 5-brolnodeoxyuridine for 2.5 min and the isolated liver nuclei were incubated with E3HITTP for lO min (incorporated of 13oo nucleotides). After incubation, the DNA was isolated, half was sheared in a Virtis homogenizer (17 S, 5600 nucleotides), the remainder was additionally broken by sonic oscillation (8 S, 900 nucleotides), and each preparation was heat denatured and centrifuged in an equilibrium gradient of neutral CsC1. After shearing in the homogenizer, the deoxynucleotides incorporated in vivo and in vitro were still in association (Fig. 2A) but, after greater shear, as can be seen from Fig. 2B, this was no longer the case. Thus, the deoxynueleotides incorporated in vitro could not have been admixed with the 5bromodeoxyuridine-labeled DNA as might be expected from a process of excision and repair.

* T h e c a l c u l a t i o n w a s b a s e d on t h e 9-fold i n c r e a s e in t h e a d d i t i o n a l c o u n t s i n c o r p o r a t e d b y t h e n u c l e i b e t w e e n I a n d 3o m i n of i n c u b a t i o n in m i x t u r e s t h a t c o n t a i n e d ? H ] T T P a n d on t h e a s s u m p t i o n t h a t 2oo n u c l e o t i d e s were a d d e d to e a c h g r o w i n g s t r a n d d u r i n g t h e fi rs t rain.

Biochim. Biophys. Acta, 287 (1972) 28-37

D N A MADE BY ISOLATED NUCLEI

35

Elongation ol both D N A strands To determine whether the isolated nuclei can elongate b o t h D N A strands, liver D N A was doubly-labeled in the rat, with E14C~thymidine for a long period a n d with [SHlthymidine for a brief time. The radioactive nuclei were isolated a n d allowed to form D N A for 30 rain in a m i x t u r e in which T T P was replaced with u n labeled 5-bromo-2'-deoxyuridine triphosphate. Analysis of the D N A in a gradient of CsC1 showed t h a t m u c h of the ~H-labeled D N A was denser t h a n the long-labeled D N A (Fig. 3A). To a p p r o x i m a t e the percentage of dense 3H-labeled DNA, as shown in the figure, the curve for long-labeled D N A was normalized so t h a t its light side coincided with the light side of the 3H peak. According to this procedure, 7 ° % of the 3Hlabeled D N A was denser t h a n the long-labeled DNA.

3H

1140 3H/

251

14C

250

25

35

45

"--

O

0

--

25

35

45

D

:i<;

"L

ZN

//

;% \

FRACTION NUMBER

Fig. 3. Density gradient analysis of DNA labeled with [SH]thymidine in vivo and 5-bromodeoxyuridine triphosphate in vitro. Each rat was partiallyhepatectomized and labeled with [t4C]thymidine (0.8/2Ci, 2 h). For A and B, the animal was given 2oo/~Ci of [SH]thymidine in the mesenteric vein and the liver was removed after 1.5 min. Nuclei were isolated and incubated for 3° min in the complete test mixture (containing o.18 M Tris buffer, (pH 8.2), EGTA, dextran and cadaverine) but TTP was replaced with unlabeled 5-bromodeoxyuridine triphosphate (o.o85 mM). After incubation, the DNA was purified with 2 M NaC1 (ref. 12) and it was then sheared in a Virtis homogenizer (A) as for Fig. 2A or additionally with sonic oscillation (B) as for Fig. 2B. For C and D, the laC-labeled nuclei were incubated for 3° rain in a mixture that contained [3H]dATP (o.o16 mM, 0. 5 Ci/mmole) and, in place of TTP, 5-bromodeoxyuridine triphosphate (o.o85 raM). The DNA preparations were sheared by homogenization (C) or by homogenization and sonic oscillation (D). Centrifugation in an equilibrium gradient of CsC1, collection of the fractions, and counting were as for Fig. 2. O - O , 8H; 0 - 0 , 1~C. The lowest curve in A ( O - ~ ) describes the values for long-labeled DNA that were normalized so that the light side of the peak could be superimposed on the light side of the 3H peak. Density decreases from left to right. The possibility was considered t h a t some of the radioactive, dense D N A of Fig. 3A h a d been made from L3HlTTP t h a t r e m a i n e d in the nuclei d u r i n g the isolation procedure a n d was t h e n incorporated in vitro along with the 5-bromodeoxyu r i d i n e triphosphate. No increase in the r a d i o a c t i v i t y of the nuclei was detected d u r i n g i n c u b a t i o n , however, and, in addition, the dense a n d SH-labeled DNAs could be almost completely separated from each other b y a d d i t i o n a l l y shearing the D N A with sonic oscillation (Fig. 3B). As controls for Figs 3A a n d 3B, nuclei t h a t had n o t been labeled with 3H

Biochim. Biophys. Acta, 287 (I972) 28-37

36

w.E. L','NCHel al.

in vivo were allowed to incorporate simultaneously 5-bromodeoxyuridine triphosphate

and [3H]dATP and the DNA was purified and subjected to the usual shear (Fig. 3C) or to a greater than usual shear (Fig. 3D). The figure shows that, with simultaneous labeling, the degree of shear had little influence on the banding pattern of the doubly labeled DNA. Since more than half of the nascent DNA made in vivo became dense during incubation of the nuclei with 5-bromodeoxyuridine triphosphate, it would seem that both DNA strands were elongated in vitro. The results were also taken to indicate that the isolated nuclei were able to advance most of the points that were growing i1~ vivo.

DISCUSSION

DNA suffers a rapid reduction in size during incubation of liver nuclei at 37 ~C. The Ca 2+ binder, EGTA, and various cations (Na +, K +, Li +) can together prevent the hydrolysis of DNA. Effective levels of the cations depress the incorporation of [3H]TTP (o.I M NaC1 causes about a 7 ° °/o inhibition) but levels of Tris or morpholinopropane sodium sulfonate (0.2 M) that reduce DNA breakdown do not inhibit deoxynucleotide incorporation. The concentration of buffer is of great importance in determining the kinetics and the nature of the reaction that the liver nuclei carry out, particularly in the absence of EGTA. Thus, with o.oi M Tris and no EGTA, the incorporation of FaH~TTP into DNA proceeds almost linearly for at least an hour but the process does not involve an elongation of the DNA chains that were growing in vivo. Nearly all of the radioactivity is associated with the bulk of the DNA rather than with the strands that were growing in the animal. EGTA not only slows the breakage of DNA but increases the initial rate of DNA synthesis by the isolated nuclei. Both effects of the chelating agent may stem from an inhibition of a Ca2+-dependent enzyme, probably an endonuclease. Of a large number of substances tested, only the diamine, cadaverine, and high molecular weight compounds, such as dextran and Ficoll, stimulate the activity of the isolated liver nuclei after the initial 5-min period of incubation. The exact means by which these compounds act is not known. Their roles would seem to be related to the nuclear membrane and they may reduce leakage from the nucleus of components of the replicative system. Cadaverine has been shown to help to preserve the morphology of isolated liver nuclei and to stimulate their rate of RNA synthesis ~ and Ficoll has been used to preserve the structural integrity of isolated chloroplasts 14'~5. Even with the improved test mixture, the rate of deoxynucleotide incorporation falls markedly after the first few minutes of incubation. The cause of the rate change is not yet known. It does not result from the exhaustion of a component of the reaction mixture or from the accumulation of an inhibitor. Fresh nuclei incorporate I~HITTP equally well in a newly prepared mixture and in a mixture that has been freed of nuclei after 3o min of incubation. Nor is it due to the completion of the portion of the overall process of DNA synthesis that the nuclei can carry out. If this were the case, it would be expected that the kinetic change would be delayed when the initial reaction rate is reduced. In fact, the kinetic patterns of incorporation Bzochi~. Biophys. Acta, 287 (1972) 28~37

DNA

MADE BY ISOLATED NUCLEI

37

are similar over a range of incubation temperatures t h a t give a 4-fold difference in initial reaction rates. The relationship between the rates of DNA formation b y liver nuclei in vivo and in vitro has been measured as 7 to I. This relationship is in good agreement with a calculated ratio. If the length of the replicative period for liver nuclei in vivo is taken to be 8 h (ref. I6), it follows that in 5 rain about IO/~g of DNA must be formed per mg of DNA. This is equivalent to the incorporation of 7.5 nlnoles of TTP. Isolated regenerating liver nuclei incorporate about o.15 nmole of T T P per mg of DNA in 5 rain but only about 15 % of the nuclei 1 account for all the incorporation. Thus, the corrected value is I nmole or 15 % of the rate in vivo. These calculations should apply even if the rate of DNA synthesis b y a single nucleus in vivo is not linear throughout the S-period T M since the population of isolated nuclei might be expected to reflect the same perturbations as were taking place in the animal. The present experiments provide additional evidence that the isolated liver nuclei can, under proper conditions, elongate the DNA chains that were growing in the animal. We have also found that the product of the nuclei behaves as doublestranded DNA on a column of hydroxylapatite 19,2°. It seems highly likely that the process in vitro is replicative in nature and that it is carried out in the same way as in vivo.

ACKNOWLEDGEMENTS

This work was supported b y grants from the American Cancer Society and the National Cancer Institute.

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