DNA nucleotidyltransferase activity of the developing rat brain

34

BIOCHIMICAET BIOPHYSICAACTA

BBA 96505

DNA N U C L E O T I D Y L T R A N S F E R A S E A C T I V I T Y OF T H E D E V E L O P I N G RAT BRAIN

JEN-FU CHIU AND S. C. SUNG Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver 8 (Canada)

(Received November ioth, 1969)

SUMMARY

DNA polymerase (deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) activity in soluble extracts from developing rat brain has been studied. The DNA polymerase from infant rat brain required the presence of template DNA, Mg 2+ and all four deoxynucleoside triphosphates. The activity was enhanced b y the addition of dithiothreitol. In developing rat brain tile DNA polymerase activity of the cerebellum was, depending upon age, 20-50 times more than that of the cortex. The activity in the cerebellum peaked at around 6 days of age and then dropped during maturation. The activity in the cortex was highest immediately after birth and went down gradually. The activity was higher with heat-denatured DNA as a template than with native DNA except at the older ages where the activity seemed to be higher with native DNA. Spermidine at a concentration of I mM stimulated the activity of DNA polymerase but at a concentration of 3 mM or higher it inhibited the activity.

INTRODUCTION In the adult brain there appears to be little or no DNA synthesis, but considerable DNA synthesis seems to take place in the brain of young animals 1-3. Although the content of DNA in the brain during its development has been studied 4-1°, very little is known concerning the regional changes in the enzymes involved in DNA synthesis during brain maturation. Recently we studied the in vitro incorporation of [14Clthymidine into DNA by various regions of the infant rat brain 2. In this previous investigation we found that the in vitro DNA synthesis in cerebellum was lO-2O times that in other regions of the brains from 6-day-old rats and the DNA synthesis in cerebellum peaked at about 6 days after birth and decreased thereafter to 18 days. However, DNA synthesis is achieved through a number of steps when the incorporation of [14CIthymi dine into DNA is measured. Now the various enzymes involved in DNA synthesis have been studied and, in this paper, the characteristics of DNA polymerase (deoxynucleoside triphosphate : DNA deoxynucleotidyltransferase, EC 2.7-7.7) of infant rat brain and the effect of spermidine thereupon are described. Bioehim. Biophys. Acta, 2o9 (197o) 34-42

DNA

NUCLEOTIDYLTRANSFERASE IN DEVELOPING BRAIN

35

M A T E R I A L S AND METHODS

Materials IMe-3H]Thymidine triphosphate was purchased from New England Nuclear. Unlabelled deoxynucleoside triphosphates, Cleland's reagent (dithiothreitol) and spermidine were obtained from Calbiochem and calf thymus DNA was from Worthington Biochemical Corporation. DNA was dissolved in o.oi M NaC1 at a concentration of 2 mg/ml and heat-denatured DNA was prepared by keeping the DNA solution at IOO° for IO rnin and cooling it rapidly in an ice bath. Sephadex G-Ioo was a product of Pharmacia (Canada).

Animals Newborn, infant and adult rats of Wistar strain were obtained from the Vivarium of this university.

Enzyme preparation Animals were decapitated and the cerebellum and cortex of each brain removed immediately with sharp pointed forceps or spatula. Cerebellum and cortex were homogenized with IO vol. and 5 vol., respectively, of ice-cold distilled water, followed b y centrifugation at 34 800 ×g for 60 min at o °. The supernatant (3-5 mg protein per ml) was used as the source of enzyme.

Assay o/DNA polymerase activity The assay measures the initial rate of the incorporation of [3HIdTTP into acidinsoluble product. The reaction mixture, in a total volume of 0.4 ml contained 20 /,moles of Tris-HC1 buffer (pH 7.4), 2/*moles of MgC12, 2/,moles of dithiothreitol, 20 nmoles each of dATP, d G T P and dCTP with 12.8 pmoles of E3H~dTTP (15. 7 C/mmole), 20/,g of native or heated calf thymus DNA and 0.05 ml of cerebellar extract or o.I ml of cortical extract. After incubation for 30 rain at 37 ° the reaction was terminated by the addition of 2.5 ml of IO °/o trichloroacetic acid at o °. The trichloroacetic acid-insoluble precipitate was washed 3 times with 5 ml each cf 5 % trichloroacetic acid. It was then dissolved in 0.2 ml of Hyamine (Packard) and mixed with io ml of scintillation solution containing 15 g of 2,5-diphenyloxazole (PPO), 15o mg of 1,4-bis-(5-phenyloxazolyl-2)benzene (POPOP) and 24 ° g of naphthalene ill I 1 each of toluene, dioxane and 95 % ethanol. After standing overnight at 4 °, radioactivity was measured in a Nuclear-Chicago Mark I liquid scintillation system. One unit of DNA polymerase activity was defined as the amount of enzyme required to convert i pmole of [3HIdTTP into the acid-insoluble product in 30 rain under the condition of assay described above.

Other methods ATPase activity was assayed b y the appearance of Pt in a reaction mixture (0.42 ml) containing 20/,moles of Tris buffer (pH 7.4), 2/,moles of MgC12, 2.52/,moles of dATP, 33.6/,moles of NaC1, I6.8/,moles of KC1 and o.I ml of brain homogenate or extract. After 20 rain incubation at 37 ° the reaction was stopped by the addition of 2.5 ml of IO % trichloroacetic acid. After removing the precipitate b y centrifugation the supernatant was analyzed for P~ by the method of GOMOR111 with some modification. Protein was estimated according to the method of LowRY et al. TM with bovine plasma albumin (Armour) as a standard. Biochim. B i o p h y s . Acta, 209 (197 ° ) 3 4 - 4 2

36

J - F . C H I U , S. C. S U N G

RESULTS

Requirement/or DNA polymerase D N A polymerase from infant brain showed properties similar to the enzymes from other organs. As shown in Table I D N A polymerase activity of Ic-day-old rat cerebellum required the addition of template D N A and Mg 2+. The activity was enhanced by the addition of dithiothreitol and by the presence of all four deoxynuTABLE

I

REQUIREMENTS FOR BRAIN D N A POLYMERASE Extract

( o . I S m g p r o t e i n ) f r o m c e r e b e l l u m of i o - d a y - o l d r a t s w a s u s e d as e n z y m e s o u r c e

Assay system

[aH]d'l'~l'P incorporation (coz~nts/n~inper assay)

7"emplate: Heated Complete system -- D N A dATP, dGTP, dCTP --dATP - dGTP -- dCTP --MgC12 -- D i t h i o t h r e i t o l -- Enzyme E n z y m e h e a t e d i o m i n a t IOO °

DAL4

5 oI 46 IOO I35 149 I ~5 96 167 49 49

Native DNA 453 46 9i 155 199 2o2 93 22o 23 23

cleoside triphosphates. With the omission of unlabelled deoxynucleoside triphosphates the incorporation of [3H]dTTP was only 2o % of that obtained in the complete system where all four nucleoside triphosphates are present, indicating primarily replicative rather than a terminal addition activity. The activity with heat-denatured D N A was somewhat higher than that with native DNA. Mg 2+ was essential for the incorporation reaction with an optinmm concentration at approximately 5 mM as shown in Fig. I. / 1200

/

,.~--~-~. /

130C

1500

J"

/

/

~00

/P",

,

"E 1000

~60C

500

\ \x

//

\~-.. c

~ 80C 2

\\4

,

/

.o.

/

/

,/"

2O0 1

2

3

4

5

5

7

62 616 710 7'4 7.'8 812 8'.5 9'0 9'4

8

pH

MgC [2 ( m M )

F i g . I. T h e effect of M g "+ o n c e r e b e l l a r D N A p o l y m e r a s e DNA as a template; 0- - -I, native DNA. F i g . 2. T h e effect of p H o n t h e D N A • - - -0, native DNA.

Biochim. Biophys. Acta,

of i o - d a y - o l d r a t brain. / x - A ,

polymerase activity.

2 0 9 (197o) 3 4 - 4 2

A-/~,

heated DNA

heated

as a t e m p l a t e ;

DNA

37

NUCLEOTIDYLTRANSFERASE IN DEVELOPING BRAIN

Properties o~ the enzyme The effect of p H on the DNA polymerase activity is shown in Fig. 2. Maximal activity was obtained around p H 8.6. The time-course of the incorporation reaction is shown in Fig. 3. An incubation time of 30 min was chosen for assay so as to be well within the period of linearity. The incorporation of ['~H]dTTP into DNA by crude enzyme extract from cerebellum was proportional to the enzyme concentration up to 54 °/~g of enzyme protein (Fig. 4)2000

f~

1500 2000

E/

1000

'=- 1500

(J

100C 50O

,/¢1,'/

u

30

60 90 Time(rain)

50C

120

02

(~4 06 08 Pr otei n( mg )

Fig. 3. T i m e - c o u r s e of t h e i n c o r p o r a t i o n of [aH]dTTP . A - & , h e a t e d D N A ! ' a s a t e m p l a t e ; 0 - - - 0 , n a t i v e DNA. Fig. 4. T h e l i n e a r i t y of t h e i n c o r p o r a t i o n r e a c t i o n w i t h e n z y m e c o n c e n t r a t i o n . E x t r a c t of cereb e l l u m from i o - d a y - o l d r a t b r a i n was used as e n z y m e source a n d h e a t - d e n a t u r e d D N A us e d a s the template.

The DNA polymerase activity in the cerebellar extract was rather unstable. As shown in Fig. 5, the activity of the crude enzyme extract dropped to about 71 ~/o 08 0.7 100

_75 o

i

0.6

12~

~0.5

1000

~0~

8o0

o 50 o4

03

25 1

2

3

400

~!

01

Oays at 0 °

600 § C>

i

o2

~ ,,t~ .

. 10.

.15 . 20 Fract ion No

200 25

Fig. 5. The s t a b i l i t y of c e r e b e l l a r D N A p o l y m e r a s e a c t i v i t y k e p t a t o - 4 ° for d a y s as i n d i c a t e d . 0-0, e n z y m e e x t r a c t alone; × - - - × , e x t r a c t k e p t in t h e pre s e nc e of 5 mlV~ d i t h i o t h r e i t o l . A c t i v i t y was m e a s u r e d w i t h h e a t e d D N A as a t e m p l a t e . Fig. 6. C h r o m a t o g r a p h y of c r u d e c e r e b e l l a r D N A p o l y m e r a s e on S e p h a d e x G - i o o . E x t r a c t (cont a i n i n g 5.2 m g of p r o t e i n in 1. 7 ml) from c e r e b e l l u m of i o - d a y - o l d r a t b r a i n w a s a p p l i e d t o a c o l u m n of S e p h a d e x G - i o o (2.o c m x 23 cm, e q u i l i b r a t e d w i t h o.005 M Tris-HC1 buffe r (pH 7.4)) a n d w a s e l u t e d w i t h t h e s a m e buffer. F r a c t i o n s of 3 m l e a c h w e r e c ol l e c t e d a t a flow r a t e of a b o u t 9 m l/h. E a c h t u b e w a s m e a s u r e d for t h e a b s o r b a n c e a t 28o a n d 26o n m a n d t h e n 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 . • -., A280 nm; A - a , i n c o r p o r a t i o n ( c o u n t s / m i n ) of [ 3 H ] d T T P w i t h h e a t e d D N A as a t e m p l a t e : O - - - 0 , i n c o r p o r a t i o n w i t h n a t i v e D N A as a t e m p l a t e .

B i o c h i m . B i o p h y s . Acta, 2o9 (197 ° ) 3 4 - 4 2

38

J-F. CHIU, S. C. SUNG

of the original after I day at o-4 ° and to 42 % after 2 days. The activity remained at this level for a few days. This loss of activity was slightly lessened b y the addition of dithiothreitol (5 raM) during storage.

Column chromatography o/cerebellar DNA polymerase on Sephadex G-xoo When the crude cerebellar extract was passed through a column of Sephadex G-ioo two main peaks of A280 nm were observed. The A2s0 nm/Aee0 am ratio of the first peak was about 0. 9 in Fraction 5 (Fig. 6) and I . i in Fraction 9. The second peak showed much higher absorbance at 260 nm than at 280 nm, e.g. the A280 .m/A26 onm ratio in Fraction 17 was 0.5, indicating the presence of nucleic acid or nucleotide. As shown in Fig. 6 two peaks of DNA polymerase activity were observed. The first peak of enzyme coincides with the first protein peak, having a strong DNA polymerase activity with a preference for heat-denatured DNA as a template. The second enzyme peak (Fraction 9), found between the two major protein peaks, showed much less activity but exhibited a preference for native DNA instead of heated DNA as a template. DATA polymerase activity in developing rat brain A study of cerebellar and cortical DNA polymerase activity in rats of 2, 6, IO and 16 days of age (Table II) indicated that the activity in the cerebellum was

TABLE

II

D N A POLYMERASE ACTIVITY OF RAT BRAIN AT DIFFERENT AGES A c t i v i t y w a s e x p r e s s e d as u n i t s / m g p r o t e i n a n d v a l u e s a r e m e a n s ~=S.D. f r o m t h r e e d e t e r m i n a tions.

Age (days)

Brain region

Activity (units~rag protein)

2

Cerebellunl Cortex

2.57 ~ o . o 9 0.094 ± 0.002

1.54 ~ o . I 6 0.069 ~ 0.006

1.67 1.36

6

Cerebellum Cortex

3.52 ± o . 1 2 o.o64-5= 0.003

2.43 ~ o . o 9 0.058 ± o.oi

1.45 I. io

io

Cerebellum Cortex

2.30 -co.I 3 0.048 ± 0.004

2.14 ± o . 1 3 0.055 ± 0.005

i.o 7 0.87

16

Cerebellum Cortex

0.79 ~-o.o7 0.043 J_ 0.003

1.14 ~ o . o 4 0.065 ± 0.003

0.69 0.66

Template: Heated DNA (a)

Ratio (a)/(b)

Native DNA (b)

always higher than in the cortex with the greatest difference (5o-fold) occurring at about 6 days of age coincident with the peak in cerebellar activity. The cerebellar activity at 16 days of age was only about 28 °/o of that at 6 days. The cortical activity was the highest immediately after birth and went down gradually during maturation. The cerebellar polymerase of newborn rats showed a preference for heat-denatured DNA as a template, but in older rats there was a progressive shift towards native DNA which was the preferred template at 16 days of age. Biochim. Biophys. Acta, 2o9 (197 o) 34-42

DNA

NUCLEOTIDYLTRANSFERASE IN DEVELOPING BRAIN

39

Inhibitory/actor When brain extracts from different ages were mixed, an inhibitory factor was noticed in the extract from older rats. Table I I I shows that when extract of cerebellum from I7-day-old or adult rats was added to the extract from 4-day-old rats the activity observed (a) was much less than the summation (b). The possible inhibitory factor in adult brain was found to be heat-labile and did not lose activity after dialysis. In order to test whether this apparent inhibitor was an enzyme which destroyed one of the reaction components, ATPase activity was measured with d A T P as the substrate. There was practically no detectable ATPase activity in the cerebellar extract, though crude eerebellar homogenate contained very high ATPase activity. TABLE III THE EFFECT OF MIXING EXTRACT FROM DIFFERENT AGES P r e p a r a t i o n s I, IX a n d I I I were c e r e b e l l a r e x t r a c t fro m 4-day-old, I 7 - d a y - o l d a n d a d u l t rats, respectively.

Preparation"

I II III

t+II I+III

Activity (counts/rain)

Ratio (a)/ (b)

Observed (a)

Expected (b)

653 142 77 6o 4 467

795 73 °

o.76 o.64

* A b o u t 0.2 nag of p r o t e i n in each p r e p a r a t i o n .

E//ect o/ spermidine on DNA polymerase SHIMIZU et al. 13 observed that the changes in the concentration of polyalnines in developing mouse brain closely paralleled the changes occurring in the DNA concentration. The developmental pattern of nucleic acid is also similar to that of polyamines in developing chick embryo 14. Furthermore, spermidine has been shown to be mandatory for substantial RNA synthesis by rat brain nuclei ~5. The effect of spermidine on DNA polymerase from developing rat brain was, therefore, investigated and the results are summarized in Table IV. At a concentration of 0. 5 mM spermidine activated the DNA synthesis at all age groups and at a concentration of I mM higher activation was observed with Io-day-old rat brain. Generally the activity with native DNA as a template was stimulated more than the activity with heat-denatured DNA as a template. At a concentration of 3 mM of spermidine, inhibition was observed with 2-day-old rats, the inhibitory effect varying over the range 20-60 °/o. When the concentration of spermidine was increased to higher than 3 raM, DNA polymerase activity from all ages was strongly inhibited.

DISCUSSION

The activity of DNA polymerase in soluble extracts from developing brain described here shows many similarities with other animal DNA polymerase 3A6,17. Th~ Biochim. Biophys. Acta, 2o9 (197o) 34-4~

I

%9 .q © v

TABLE

IV

(3)

~O8 ~_ 3 (3)

I.O

4 0 ~' 2 °

I 2 0 - K 7 (3)

3 .0

i00

0"5

30±~I5

II3~I6

I4I~I3

I00

(3)

(4)

(4)

8 (6)

96~I3

(5)

I 2 2 E K 1 6 (5)

I2I-~

I00

Heated D N A

Heated D N A

Native D N A

go days

2 days

Age o/rats:

0

(raM)

Spermidiue

(5)

(5)

73-4215 (5)

I9I~I5

I5I~-I9

IO0

Native D N A

95-~

(5)

(6)

8 (4)

I25~215

II9zKI6

IO0

Heated DN,4

±6 days

(6)

7 8 - ~ 9 (4)

I6OZK22 (4)

I55~20

IO0

Native D N A

C e r e b e l l a r e x t r a c t s f r o m d i f f e r e n t a g e s of r a t s w e r e u s e d a s t h e s o u r c e o f e n z y m e w i t h e i t h e r h e a t - d e n a t u r e d D N A o r n a t i v e D N A a s t h e t e m p l a t e . S p e r m i d i n e w a s a d d e d a t t h e f i n a l c o n c e n t r a t i o n s a s i n d i c a t e d . V a l u e s a r e m e a n s ± S . D . a n d t h e a c t i v i t y w i t h o u t a d d i t i o n of s p e r n l i d i n e w a s t a k e n as i o o . N u m b e r of d e t e r m i n a t i o n s a r c i n p a r e n t h e s e s .

EFFECT OF SPERMIDINE ON D N A POLYMERASE FROM BRAIN

©

0o ¢b

4~ o

DNA

NUCLEOTIDYLTRANSFERASE IN DEVELOPING BRAIN

4.1

enzyme requires the presence of template DNA, Mg 2+ and all four deoxynucleoside triphosphates. Like most of the animal DNA polymerases 1~,17 the enzyme from developing rat brain prefers the heat-denatured DNA as a template, except at older age (e.g. at 16 days of age, see Table II) where the activity seems to be higher with native DNA as a template. Furthermore, as shown in Fig. 6 there seems to be two kinds of DNA polymerase in cerebellar extract; one prefers the heated DNA as a template and the other prefers native DNA as a template. Preparations of DNA polymerase from regenerating rat liver is, rat liver mitochondria 19 and sea urchin embryos e° have been reported to show a strong preference for native DNA as a template. Investigation is now under progress to see if the activity ratio of ttle first enzyme peak (which prefers heated DNA) (Fig. 6) to the second enzyme peak (which prefers native DNA) will increase with 2-day-old rat brain and decrease with I6-dayold rat brain since preference toward heated DNA appeared to shift progressively towards native DNA as a preferred template during maturation. In accordance with our previous study on the incorporation of [14C~thymidine into developing rat brain DNA 2, the activity of DNA polymerase in cerebellum is 2o-50 times higher than that in cortex depending on the age. The activity in cerebellum attains at the m a x i m u m level at around 6 days of age and then decreases rapidly during maturation to adult where practically no activity could be detected. Polyamines such as spermidine and spermine have been known to be widely distributed in biological material and be involved in various biological systems ~1. Polyamines bind to nucleic acids 21, form complexes with denatured DNA 22, and synthetic polynucleotides 23 and increase the denaturation temperature of native DNA ~4. Since spermidine at a concentration of i mM inhibits rat brain deoxyribonuelease both at p H 7.4 and p H 8. 9 by 70-80 % (our unpublished observation), the stimulation of DNA synthesis b y spermidine (Table IV) m a y be due to the protection of DNA by spermidine from the concomitant deoxyribonuelease activity. However, the mechanism by which spermidine stimulates DNA synthesis at low concentration (I raM) and inhibits at high concentration (3 mM or higher) and affects, either stimulates or inhibits, more the activity with native DNA as a template than that with heat-denatured DNA is obscure. ACKNOWLEDGMENT

This work was supported by a grant from the Medical Research Council of Canada. REFERENCES i 2 3 4 5 6 7 8 9

D. H. ADAMS, Biochem. J., 98 (1966) 636. S. C. SUNG, Can. J. Biochem., 47 (1969) 47F. L. 3/IARGOLIS, dr. Neurochem., 16 (1969) 447. I. LESLIE AND J. N. DAVlDSON, Biochim. Biophys. ,4eta, 7 (1951) 413 . A. V. 1DALLADIN,in H. WAELSCH, Biochemistry o[ the Developing Nervous System, A c a d e m i c Press, N e w Yo rk , 1955, p. 177. J. SZEPSENWOL, J. MASON AND M. E. SHONTZ, JJm. J. Physiol., 18o (1955) 525 • L. L. UZMAN AND M. K. RUMLEY, J. Neurochem., 3 (1958) 17°. P. 3/[ANDEL, H. REIN, S. HARTH-EDEL AND R. Y[ARDELL, in D. RICHTER, Comparative Neurochemistry, lVfacmillan, N e w York, 1964, p. 149. M. •VINICR AND A. NOBLE, Develop. 13iol., 12 (1965) 451-

Biochim. B i o p h y s . Acta, 209 (197 ° ) 34-42

42

J-F. CHIU, S. C. SUNG

i o S. S. OJA, Ann..Acad. Sci. Fennicae, Ser. A V, 125 (1966) 17. i i G. GOMORI, J. Lab. Clin. Zgled., 27 (1942) 955. 12 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND 1~. J. RANDALL, J. Biol. Chem., 193 ( t 9 5 i ) 265. 13 H. ~qHIMIZU, Y. I~AKIMOTO AND I. SANO, Nature, 207 (1965) 1196. 14 C. M. CALDARBRA, B. t3ARBIROLI AND G. MORUZZI, Biochem. J., 97 (1965) 84. 15 O. I{. DUTTON AND H. R. iVfAHLER, J. Neurochem., 15 (1968) 765 . 16 F. J. BOLLUM, in J. N. DAVlDSON AND W. E. COHN, Progress in Nucleic .4cid Research, Vol. i, A c a d e m i c Press, N e w York, 1963, p. i. 17 H. M. I(EIR, in J. N. DAVlDSON AND ~,V. E. COHN, Progress in Nucleic Acid Research and Molecular Biology, Vol. 4, A c a d e m i c Press, N e w York, 1965, p. 81. 18 J. T. BELLAIR, Biochim. Biophys. Acta, 161 (1968) 119. 19 G. F. KALF AND J. J. CH'IH, J. Biol. Chem., 243 (1968) 4904 . 20 L. A. LOEB, J. Biol. Chem., 244 (1969) 1672. 21 H. T~.BOR AND C. W. TABOR, Pharmacol. Rev., 16 (1964) 245. 22 O. FELSENFELD AND S. L. HUANG, Biochim. Biophys. Aeta, 51 (1961) 19. 23 S. L. HUANG AND G. FELSENFELO, Nature, 188 (196o) 3Ol. 24 H. TABOR, Biochemistry, I (1962) 496.

Biochim. Biophys. Acta, 2o9 (197 ° ) 34 42