Mutation Research, 237 (1990) 65-73

65

Elsevier MUTAGI 090~3

Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development F e d e r i c o F o c h e r 1, P a o l o M a z z a r e l l o 2, A n n a l i s a V e r r i 1, U l r i c h H t i b s c h e r 3 a n d Silvio S p a d a r i 1 1 lstituto di Genetica Biochimica ed Eooluzionistica, CNR, 1-27100 Paoia (Italy), 2 Clinica Neurologica I, Unioersit,~ di Milano, 1-20142 Milan (Italy) and J Department of Pharmacology and Biochemistry, University of Ziinch-lrchel, CH-8057 Ziirich (Switzerland)

(Received 21 June 1989) (Revision received 5 December 1989) (Accepted 18 December 1989)

Keywords: DNA polymerase fl; Uracil DNA-glyeosylas¢; Nucleoside diphosphokinase; dUTPase; DNA repair in neurons

Summary We have shown that DNA polymerase fl, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between d T r P and dUTP, having the same K m for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase ftmetions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.

Among the many enzymes and factors involved in the process of DNA replication and repair, a central role is played by DNA polymerases. Mammalian cells possess 4 DNA polymerases named or, r , "t and ~(for recent reviews see Kaguni and Lehman, 1988; Wilson et al., 1988; Fry and Loeb, 1986; So and Downey, 1988, respectively). DNA polymerases a and 8 appear essential for nuclear DNA replication (Hiibscher et al., 1978; Spadari

Correspondence: Silvio Spadafi, Istituto di Genetica Biochimica ed Evolmionistiga, CNR, Via Abbiategrasso, 207, 1-27100 Pavia (Italy).

et al., 1982, 1988, 1989), DNA polymerase fl is mainly associated with DNA repair (Hiibscher et al., 1979) and DNA polymerase y is the mitochondrial DNA-replicating enzyme (Hiibscher et al., 1979). Adult neurons lack DNA polymerases a and ~ (Hiibscher et al., 1979; Spadari et al., 1988) and this correlates well with the fact that at birth neurons stop DNA replication, they cease to divide and once destroyed Other by aging or diseases, they cannot be replaced. Their nuclei contain exclusively DNA polymerase fl (Hfibscher et al., 1979; Waser et al., 1979). This enzyme is highly error-prone in selecting the complementary

0921-8734/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

66 base during reparative DNA synthesis (Kunkel and Loeb, 1980, 1981) and its low level of fidelity remains substantially unchanged during the neuron life span (Subba Rao et al., 1985). There are some suggestions that damage to DNA might be an important component in the biology of aging (for a review see Kirkwood, 1989) and in some degenerative disorders of the nervous system (Lewis et al., 1981; Mann and Yates, 1974; Mazzarello and Poloni, 1988). In this paper we devote our attention to the possibility that uracil incorporation into DNA by DNA polymerase fl can occur in adult neurons during DNA-repair synthesis. If not fully repaired a significant replacement of thymine by uracil in DNA could affect the recognition of substrate nucleotide sequences by enzymes a n d / o r by regulatory DNA-binding proteins (Weiss and E1-Hajj, 1986). An enzyme essential for the removal of uracil from DNA is uracil DNA-glycosylase, which catalyzes the specific removal of uracil from DNA by cleaving the N-glycosylic bond linking the base to the deoxyribose phosphate backbone. We have therefore investigated: (1) the ability of DNA polymerase fl to incorporate dUTP into DNA; (2) the perinatal levels of nuclear uracil DNA-glycosylase and (3) the perinatal levels of dUTPase and nucleoside diphosphokinase (NDP kinase), enzymes that modulate the pool size of dUTP. High levels of dUTP could in fact favor the misincorporation of uracil into DNA by DNA polymerases (Goulian et al., 1980; Tye et al., 1977).

Whatman. Hydroxyapatite was prepared according to Bernardi (1971). DNA polymerase I (Klenow fragment) was purchased from Boehringer. All other chemicals and reagents were purchased from local suppliers.

Animals and isolation of neurons SIV rats of either sex were used throughout this study. Developmental stages included fetuses of 19, 20 and 21 days' gestation (the mean gestation in rats is 22 days), as wall as animals of postnatal ages 0 (newborn), 1, 2, 7, 14 and 21 days. At each developmental stage 20 animals (fetuses or born) were killed by decapitation and the forebrains were removed. Neurons from forebrain hemispheres (fetuses and postnatal stages 0-2 days) and forebrain cortex (postnatal stages 7-21 days) were obtained by the method of Kuenzle et al. (1977). The neurons were immediately frozen and kept at - 80 o C until use. Preparation of neuronal crude extracts In order to test dUTPase and NDP kinase the neurons were resuspended in 3 volumes of 10 mM Tris-HC1 (pH 7.5), 10 mM KC1, 1 mM Dq'T, 0.1 mM EDTA, 1 mM ATP, 4 mM sodium metabisulrite, 1/~M pepstatin and 1 mM PMSF. To test uracil DNA-glycosylase, found preferentially in the nuclear fraction, the previous extraction buffer was made 400 mM KC1. Sonication on ice, in a Branson sonifier at 70 watt 4 times for 4 s at intervals of 20 s, followed. The extract was centrifuged at 8000 × g for 10 min in an Eppendorf centrifuge.

Materials and methods

Materials Deoxyribonucleoside triphosphates were from Amersham ([3H]-labelled) or from Boehringer (unlabelled). Pharmacia was the supplier of heparinSepharose CL-6B. Pepstatin A and phenylmethylsulfonyl fluoride (PMSF) were from Sigma; bovine serum albumin (BSA), A grade, was from Calbioehem. Sodium metabisulfite and thin-layer chromatography (TLC) plastic sheets polyethyleneimine (PEI)-cellulose F (layer thickness 0.1 mm) were from Merck. NP-40 was from BDH and dithiothreitol (DTY) was obtained from Fluka. Phosphocellulose (Pll) and DE32 were from

Purification of DNA polymerase fl Ten rats of the SIV strain (60 days old) were killed by decapitation, the forebrains immediately removed and the cortices dissected in the cold. The 10 cortices (8 g) were resuspended in 30 ml of a buffer containing 1 mM potassium phosphate (pH 6.8), 10 mM NaC1, 0.2 mM PMSF, 1 mM DTT, 0.1 mM EDTA, 4 mM sodium metabisulrite, 1 /~M pepstatin (buffer A). After 10 rain on ice the cortices were homogenized in a Dounce homogenizer. The homogenate was then centrifuged at 7000 rpm in a Beckman JA20 rotor for 15 min in order to precipitate the nuclei. The pellet was resuspended in 30 ml of buffer A and homog-

67 enization and centrifugation were repeated as above. The pellet was then resuspended in 30 ml of a buffer containing I mM potassium phosphate (pH 7), 0.32 M sucrose, 1 mM MgC12, 0.3% NP-40, 1 mM DTT, 0.2 mM PMSF, 0.1 mM EDTA, 4 mM sodium metabisulfite and 1 pM pepstatin (buffer B). In this buffer the nuclei were homogenized and centrifuged at 10,000 rpm in the Beckman JA20 rotor for 15 min. The pellet was then resuspended in 30 ml of the following buffer: 400 mM potassium phosphate (pH 7.5), 0.3 M KC1, 1 mM DTF, 0.1~ NP-40, 1 mM PMSF, 1~ dimethyl sulfoxide, 4 mM sodium metabisulfite, 1 pM pepstatin (buffer C). After 15 min on ice the nuclei were disrupted in a Dounce homogenizer. The homogenate was centrifuged for 90 min at 25,000 rpm in a Sorvall T865 rotor. The supernatant was loaded on a DE-32 column (volume = 10 ml), previously equilibrated with 10 column volumes of buffer C, to remove nucleic acids. The flow-through and the first 10 ml of the subsequent column wash with buffer C were collected and dialyzed against the following buffer: 0.5 mM DTT, 0.2 mM PMSF, 0.15g NP-40, I/~M pepstao tin, 4 mM sodium metabisulfite (buffer D) in order to lower the ionic strength to 50 mM potassium phosphate (pH 7.5). The cloudy dialyzed solution was then centrifuged at 10,000 rpm for 15 rain and the supernatant was loaded onto a DE-32 column (volume = 5 ml). The flow-through and the first 5 ml of the subsequent colunm wash with buffer D made 50 mM potassium phosphate (pH 7.5) (buffer E), containing DNA polymerase [3, were loaded onto a phosphocellulose column (volume = 5 ml) equilibrated with buffer E. The phosphocelluiose column was washed with 4 column volumes of buffer E, 5 column volumes of buffer D made 100 mM potassium phosphate (pH 7.5), 5 column volumes of buffer D made 150 mM potassium phosphate (pH 7.5) and then the DNA polymerase [3 was eluted with 3 colunm volumes of buffer D made 350 mM potassium phosphate (pH 7.5). The pooled fractions were dialyzed against 20 mM potassium phosphate (pH 7), 50 mM KC1, 30% glycerol, 1 mM DTT, 0.5 mM EDTA, 0.1 mM EGTA, 1 pM pepstatin and 0.2 mM PMSF (buffer F) and then loaded onto a heparin-Sepharose column (volume = 0.5 ml) equilibrated with

buffer F. After 2 colunm washes at 20 mM and 300 mM KC1 in buffer F the DNA polymerase [3 was eluted with 0.8 M KC1. The pooled fractions were immediately frozen in small aliquots in liquid nitrogen and stored at - 1 9 6 ° C until use. The final preparation had a specific activity of 200 units/rag (one unit is defined as I nmole of dNTP incorporated into acid-precipitable DNA in 1 h at 37°C) and was free of dUTPase and dTTPase activities.

Purification of human DNA polymerase a, DNA polymerase [3 and PCNA-independent DNA polymerase ~ DNA polymerases a and ~ were purified from 100 g of HeLa cells through 3 chromatographic steps namely phosphocellulose, hydroxyapatite and heparin-Sepharose as outlined in Focher et al. (1988, 1989). DNA polymerase [3 was purified from HeLa cells following the method outlined above for the purification of rat neuronal DNA polymerase [3 with a few inessential modifications. DNA polymerase [3 assay The reaction mixture (25/~1) contained: 50 mM Tris-HCl (pH 8.6), 10 mM MgC12, 100 mM KC1, 1 mM DTI', 0.25 mg/ml BSA, 2 pg activated DNA, 100 pM each of dATP, dGTP and dCTP, 20 pM [3H]dTrP (250 cpm/pmole) or 20 /~M [3H]dUTP (250 cpm/pmole) and enzyme to be tested. After incubation at 37 °C for 30 rnin 20 pl of the mixture was spotted on G F / C filters (Whatman). The filters were washed 3 times in 5~ (v/v) trichloroacetic acid (TCA) for 5-10 rain and twice in ethanol. They were then dried and the acid-insoluble radioactivity was estimated by scintillation counting of the filters in 1 ml of scintillating mixture. DNA polymerase ~ assay The reaction mixture (25/~1) contained: 20 mM potassium phosphate (pH 7.2), 0.1 mM EDTA, 4 mM DTT, 0.25 mg/ml BSA, 10 mM MgC12, dATP, dGTP and dCTP each at 48 ~M, 18 ~M [3H]dTFP (250 cpm/pmole), 3/~g activated DNA and enzyme to be tested. After incubation at 37 ° C for 15 rain the acid-insoluble radioactivity was determined as described above for the DNA polymerase [3 assay.

68

DNA polymerase ~ assay The reaction mixture (25 #1) contained: 75 mM Hepes-KOH (pH 7.5), 1.25 mM DTT, 205[ glycerol, 10 mM MgC12, 0.24 m g / m l BSA, 10 mM KC1, 0.5 ~g po!]c,(dA)/oligo(dT)12_18 (base ratio 10:1), 10 #M [ H]dTTP (250 cpm/pmole) and enzyme to be tested. After incubation at 37 ° C for 15 min the acid-insoluble radioactivity was determined as described above for the DNA polymerase/3 assay. Preparation of [3H]dUMP- or [3H]dTMP-containing DNA 0.2 mg of activated DNA, prepared according to Pedrali-Noy and Weissbach (1977), was incubated in 1 ml of 20 mM potassium phosphate (pH 7), 0.1 mM EDTA, 4 mM DTT, 0.25 m g / m l BSA, 10 mM MgC12, 100 /~M each of dATP, dGTP and dCTP, 20 /~M [3H]dUTP (1140 c p m / p m o l e ) or 20 /~M [3H]dTTP (1500 cpm/pmole) and 40 units of DNA polymerase I (Klenow fragment). The incubation was carried out at 37 °C for 2 h. After incubation the solution was made 1.5 M ammonium acetate and the DNA was precipitated with 60% (v/v) isopropanol. After 1 h at room temperature the DNA was centrifuged at 12,000 rpm in an Eppendorf centrifuge for 20 min. The supernatant was removed and the pellet was carefully washed 3 times with 70% ethanol. The pellet was resuspended in 10 mM Tris-HC1 (pH 7.5) and 1 mM EDTA to a final concentration of 1 mg/rnl. The specific activity of this DNA was 40 cpm/ng. Uracil DNA-glycosylase assay The reaction mixture (25 /~1) contained: 100 mM Tris-HC1 (pH 8), 5 mM DTT, 10 mM EDTA, 500 ng of [3H]dUMP-labelled DNA (40 cpm/ng), 4 /~g of unlabdled activated DNA and partially purified neuronal crude extracts to be tested. After incubation at 37 °C for 30 rain the acid-insoluble material was determined as described above for the DNA polymerase/3 assay. In parallel, as control, the acid-soluble material was determined as follows: samples were made 1.5 M ammonium acetate and DNA was precipitated with 75 /~1 of cold ethanol. After 30 rain at --20 °C the DNA was centrifuged at 12,000 rpm in an Eppendorf centrifuge for 20 min. 80 /~1 of supernatant was

collected and the acid-soluble material was determined in a beta counter using Instagel solution. One unit of uracil DNA-glycosylase is defined as 1 nmole of uracil removed from the DNA in 1 h at 37 ° C.

dUTPase and nucleoside diphosphokinase (NDP kinase) assay The reaction mixture (25/~1) contained: 75 mM Hepes-KOH (pH 7), 1.25 mM DTT, 20% glycerol, 10 mM MgC12, 0.24 mg/ml BSA, 20 /~M [3H]dUTP (1140 cpm/pmole) and neuronal extract to be tested. After 15 min at 37°C 1 #1 of the reaction mixture was loaded on a TLC plastic sheet PEI-cellulose F. To each spot 2 /d of a solution containing dUTP, dUDP or dUMP, 20 mM each, was added in order to have optical markers at UV light (254 nm). Chromatography was performed in 0.5 M ammonium formate (pH 3.4). The sheet was then dried and the single lanes were cut into 5 pieces according to the marker positions. To the single piece, in a vial, 150/~1 of distilled water and 0.5 ml of Soluene®-350 (Packard) were added. The vials were then incubated for 1 h at 55 °C and cooled down to room temperature in darkness. 3 ml of scintillating mixture was added to the vials, whose radioactivity was finally counted in a beta counter. One unit of dUTPase or NDP kinase is 1 nmole of dUTP transformed in dUMP or dUDP, respectively, in 1 h at 37°C. Results

DNA polymerase fl incorporates dUTP into DNA more efficiently than DNA polymerases a and ~ Some studies have indicated that DNA polymerase/3 is highly error-prone in the selection of the complementary base during DNA synthesis (Kunkel and Loeb, 1980, 1981). Compared with calf thymus DNA polymerase a (error rate 1/30,000) or E. coli polymerase I (error rate 1/680,000), DNA polymerase /3 has a very low accuracy showing an error rate of 1/8000 (Subba Rao et al., 1985). Based upon the hypothesis that aging and some degenerative disorders of the nervous system may be correlated with DNA damages we tested the capacity of DNA polymerase/3, the only nuclear DNA polymerase pres-

69

O~

'

30

"

A

~

600

,

[]

B

~

× 400 ~.

20

"

i

=

~ "o

0

0

1 2 1/S, pM "~

0

3

0

1 1/S,

200

2

3

~M- 1

150

~

C

,

~o

,~

D

~ x

X

~; ~00

~;

"o

"o

0

0

100

1 2 1/S, pM - ~

3

0

0

1 2 1/S, pM ~

3

Fig. 1. Determination of K m for dTTP and dUTP ot rat neuronal DNA polymerase ~8 (A) and human DNA polymerase ~8 (B), DNA polymerase a (C) and DNA polymerase ~ (D). Enzymes were tested in their specific assay as described in Materials and methods by varying the concentration of dTTP ([3 [3) or dUTP ( • • ) . In this as well as in the following figures data are the average

of 3 determinations.

ent in adult neurons, to incorporate the wrong nucleotide dUMP into DNA. We thus evaluated the K m for dUTP and dTTP of rat neuronal DNA polymerase fl and compared them with those obtained with other DNA polymerases, namely human DNA polymerases a, fl, and & The results are reported in Fig. 1 (A-D) and show that DNA polymerase /~ either from rat neurons or from HeLa cells has identical Kr~ for d T r P and dUTP (2.5 #M for the rat and 2 #M for the human enzyme), whereas DNA polymerase a and PCNA-independent DNA polymerase ~ are more discriminating enzymes showing different K m for these substrates (11/tM dTTP and 29 ttM dUTP for DNA polymerase a and 1 /~M dTTP and 2 /~M dUTP for DNA polymerase 8). Thus in adult

neurons, whose nuclei only contain DNA polymerase/], the incorporation of dUMP into DNA is solely dependent on the relative concentration of dUTP in the cell since DNA polymerase ~ is unable to discriminate between the 2 substratcs.

Effect of Mn 2+ on d U T P incorporation into DNA by DNA polymerase fl Mn2+ reduces the fidelity of DNA polymerases by decreasing the K m of the polymerase-DNA complex for the mismatched deoxynucleotide (Goodman et al., 1983; H-Deity et al., 1984). To test the effect of Mn2+ on the incorporation into DNA of dUTP by DNA polymerase fl we first evaluated the concentrations of Mg 2+ and Mn2+ required for optimal incorporation of dUTP into

70

60 z

A

50

0

E 40 30 0

20

,,~ 10, .Z £3 0 0

I

I

I

I

5

10

15

20

25

Mg++, mM

~-

40

B

a0 t).

d •~ 20 N >" 10 ,~ z tm

0

u,O

I

0,5

I

1,0 Mn++, mM

I

1,5

2,0

Fig. 2. Effect of divalent cations on the incorporation of d U T P by D N A polymerase ft. 0.1 U of D N A polymerase fl was tested as described in Materials and methods with different concentrations of Mg 2+ (A) or Mll2+ (B).

quent mutagenic effects of these phenomena could be minimized by low intracellular levels of dUTP and by uracil DNA-glycosylase, an enzyme that catalyzes the specific removal of uracil from DNA by cleaving the N-glycosylic bond linking the base to the deoxyribose phosphate backbone. Therefore we have studied the developmental activity profiles of uracil DNA-glycosylase and of dUTPase and NDP kinase, enzymes that control the pool size of dUTP. In developing rat neurons in fact enzymes such as DNA polymerases a and ~, which play a major role in DNA replication, drop sharply in an identical pattern from a high level with the approach of the term of gestation and disappear at approximately 2-3 weeks of postnatal age (Hiibscher et al., 1977, 1979; Spadari et al., 1988). By contrast, levels of DNA polymerase /3, mainly involved in DNA repair, and of DNA polymerase 7, responsible for the replication of mitochondrial DNA, remain constant (Hiibscher et al., 1977, 1979). The enzymatic activities of uracil DNA-glycosylase, dUTPase and NDP kinase were determined in neurons from day 3 before birth up to day 21 after birth. The results are presented in Fig. 4. Fig. 4A shows a significant age-dependent decrease in the levels of uracil DNA-glycosylase

20

DNA by DNA polymerase/3. Fig. 2 shows that maximum incorporation of dUTP occurs either at 10 mM Mg 2+ or at 0.2 mM Mn 2+. However, data in Fig. 3 demonstrate that an increasing amount of Mn 2÷ has substantially no preferential effect on the selection of the nucleotide substrate by DNA polymerase/3 showing no differential K m changes between dTTP and dUTP.

Z~ 15

~

5

Z

Developmental activity profiles of uracil DNA-glycosylase, dUTPase, nucleoside diphosphokinase in nerve cells The above results indicate that DNA polymerase/3, the only DNA polymerase present in adult neuronal nuclei, can efficiently incorporate dUTP into DNA. Uracil in DNA arises also from spontaneous cytosine deamination. The conse-

0

~i

u,O

I

0,5

I

1,0 Mn++, rnM

I

1,5

2,0

Fig. 3. Effect of M n 2 + on the choice of nucleotide substrate by D N A polymerase ft. 0.03 U of D N A polymerase fl was tested at different concentrations of Mn 2+ in an assay containing 10 p M [3H]dTTP/10 p M dTTP (El t~) or 10 p M [3H]dUTP/10/~M dTTP (O O)-

71 TABLE 1

A

EFFECT OF DIFFERENT SUBSTRATES ON URACIL DNA-GLYCOSYLASE ACTIVITY FROM -2-DAY RAT NEURONS

!

0

-4

0

4

8 12 16 20 24 days

200

~

100

l

0 -4 0

~ 150

I

4

I

I

l

l

I

C

50

"~

[3H]Uracil-conhainin$ DNA [3H]Thymine-containing DNA

15.704-0.27 0.00+0.08

~ The total amount of labelled nucleotide in DNA is 22 pmole.

n a l extracts o n a D N A t e m p l a t e l a b e l l e d with [3H]thymidine. This t e m p l a t e was c o m p l e t e l y inactive in o u r a s s a y ( T a b l e 1). T h e specificity o f the uracil D N A - g l y c o s y l a s e a s s a y was f u r t h e r d e m o n s t r a t e d b y a n a l y z i n g the p r o d u c t s o f the r e a c t i o n o n T L C - P E I c h r o m a t o g r a p h y . R a d i o a c t i v i t y was fully a s s o c i a t e d w i t h uracil, as expected, a n d n o t w i t h d U M P (Fig. 5). T h e s e d a t a are in a g r e e m e n t w i t h those o f o t h e r a u t h o r s (Sirover, 1979; G u p t a a n d Sirover, 1981), w h o d e m o n s t r a t e d t h a t in n o r m a l h u m a n cells

100 i

-

~

~

ao 6O40-

~

~ z

Radioactive material removed (pmole/15 min)

8 12 16 20 24 days

-~ 100

~

Substrate a

~ . . . . . . . -4 0 4 8 12 16 20 24 days

20-

0

Fig. 4. Developmental activity profdes of uracil DNA-glycosylase (A), dUTPase (B) and NDP kinase (C) in rat neurons.

w h o s e p a t t e r n very m u c h r e s e m b l e s t h a t o f D N A p o l y m e r a s e s a a n d ~ ( S p a d a r i et al., 1988). T h e p o s s i b i l i t y that n o n - s p e c i f i c nucleases c o u l d m i m i c t h e uracil D N A - g l y c o s y l a s e activity in o u r a s s a y h a s b e e n r u l e d o u t b y testing in p a r a l l e l the n e u r o -

0

1

2 3 Fractions

4

Fig. 5. P~lio~ti~ty removed from [3H]uracil-DNA by uracil DNA-glycosylase from - 2-day rat neurons is exclusively associated with the uracil fraction. The uracil DNA-glycosylas¢ assay was performed as described in Materials and methods. After 15 rain at 37°C, 3 pl of the reaction mixture was loaded on a TLC plastic sheet PEI-cellulose F. Optical markers were added. Chromatography was performed in 0.25 M LiCL The sheet was then dried and the single lanes were cut into 4 pieces according to the marker positions.

72 uracil DNA-glycosylase activity is inducible during the cell cycle, and are supported by data which indicate a physical association of uracil DNA-glycosylase with D N A polymerase a (Seal and Sirover, 1986). As D N A polymerase fl is not able to discriminate between dTTP and d U T P and uracil DNA-glycosylase is nearly absent in adult neurons, the presence of uracil residues in D N A is directly influenced by the pool size of dUTP. Therefore we have studied the activity profiles during neuronal development of 2 enzymes which control the d T T P / d U T P ratio, namely dUTPase, which transforms d U T P into dUMP, the precursor of dTTP, and nucleoside diphosphokinase ( N D P kinase), which contributes in establishing an equilibrium between the levels of di- and triphosphate nucleosides with either a ribose or a deoxyribose configuration. Fig. 4B shows the developmental activity profile of dUTPase, an enzyme that by hydrolyzing d U T P to dUMP, which is then converted to dTMP, increases the size of the d T T P pool and drastically reduces the d U T P pool. Its specific activity (more than 100 U / m g ) is 2-3-fold higher than that observed in a parallel assay with HeLa extracts (data not shown) and slightly increases during the development of neurons. To follow the N D P kinase during neuronal development, we have tested its d U T P dephosphorylating activity (dUTP ~ dUDP) and the resuits indicate that in neurons this activity decreases during neuronal development (Fig. 4C). However, considering that (1) both dUTPase and N D P kinase activities have been determined in the same assay with [3H]dUTP as common substrate (because of the lack of commercially available [3H]dUDP), and (2) N D P kinase converts d U T P to d U D P and vice versa, giving new substrate to dUTPase (which irreversibly transforms d U T P into dUMP) and lowering its reaction product dUDP, the apparent developmental decrease in N D P kinase activity could be attributed to the increased dUTPase activity.

Concluding remarks We have shown that the K m for dTTP and d U T P of D N A polymerase /3, the only D N A

polymerase present in adult neuronal nuclei, are identical. Therefore the extent of d U M P incorporation into nuclear D N A of adult neurons is directly related to the size of the intracellular d U T P pool. In dividing neurons, incorporated uracil, either due to D N A polymerase fl or resulting from cytosine deamination, may very rapidly be excised by uracil DNA-glycosylase. In resting neurons the reduced levels of uracil DNA-glycosylase (this work) might impair the removal of uracil from DNA. However, the high levels of dUTPase throughout neuronal development could prevent an increase in the size of the d U T P pool and thus keep the frequency of uracil incorporation into D N A during D N A synthesis to low levels. One cannot rule out the possibility that some degenerative disorders of the nervous system or aging as well may at least be partially correlated with the presence or absence of enzymes that alter the normal pathways of deoxynucleoside triphosphate biosynthesis (Mazzarello et al., 1989).

Acknowledgements We would like to thank Dr. Giovanni Ciarrocchi for his helpful suggestions, stimulating discussions and critical reading of the manuscript. P.M. was a "B. Vieri" fellow of the Associazione Italiana Sclerosi Laterale Amiotrofica. This work was supported by the C.N.R. Programmi Finalizzati Biotecnologie and Chimica Fine.

References Bernardi, G. (1971) Chromatography of proteins on hydroxyapatite, Meth. Enzymol.,22, 325-327. E1-Deiry, W.S., K.M. Downey and A.G. So (1984) Molecular mechanism of manganese mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 81, 7378-7382. Focher, F., S. Spadari, B. Crinelli, M. Hottiger, M. Gassmann and U. Hiibscher (1988) Calf thymus DNA polymerase ~: purification, biochemical,and functional properties of the enzyme after its separation from DNA polymerase a, a DNA dependent ATPase and proliferating cell nuclear antigen, Nucleic Acids Res., 16, 6279-6295. Focher, F., M. Gassmann, P. Hafkemeyer,E. Ferrari, S. Spadari and U. Htibscher (1989) Calf thymus DNA polymerase 8 independent of proliferating cell nuclear antigen (PCNA), Nucleic Acids Res., 17, 1805-1821. Fry, M., and L.A. Loeb (1986) Animal Cell DNA Polymerases, CRC Press, Boca Raton, FL.

73 Goodman~ M.F., S. Keener, S. Guldotti and E.W. Branscomb (1983) On the enzymatic bases for mutagenesis by manganese, J. Biol. Chem., 258, 3469-3475. C~ullan, M., B. Bl~ile and B.Y. Tseng (1980) Methotrexate-induced misincorporation of uracil into DNA, Proc. Natl. Acad. Sci. (U.S.A.), 77, 1956-1960. Gupta, P.K., and M.A. Sirover (1981) Stimulation of the nuclear uracil DNA glycosylase in proliferating human fibroblasts, Cancer Res., 41, 3133-3136. Htibscher, U., C.C. Kuenzle and S. Spadad (1977) Variation of DNA polymerases a, fl and 3' during perinatal tissue growth and differentiation, Nucleic Acids Res., 4, 29172929. Hilbscber, U., C.C. Kuenzle, W. Limacher, P. Scherrer and S. Spadari (1978) Functions of DNA polymerase a, fl and "t in neurons during development, Cold Spring Harbor Symp. Quant. Biol., 43, 625-629. Hfibscher, U., C.C. Kuenzle and S. Spadari (1979) Functional roles of DNA polymerase fl and y, Proc. Natl. Acad. Sci. (U.S.A.), 76, 2316-2320. Kaguni, LS., and I.R. Lehman (1988) Eukaryotic DNA polymerase a-primase: structure, mechanism and function, Biochim~ Biophys. Acta, 950, 87-101. Kirkwood, T.B.L. (1989) DNA, mutation and aging, Mutation Res., 219, 1-7. Kuenzle, C.C., A. Kniisel and D. Schiimperli (1977) Isolation of neuronal nuclei from rat brain cortex, rat cerebellum and pigeon forebrain, Meth. Cell Biol., 15, 89-96. Kunkel, T.A., and L.A. Loeb (1980) On the fidelity of DNA replication: the accuracy of Escherichia coil DNA polymerase I in copying natural DNA in vitro, J. Biol. Chem., 255, 9961-9966. Kunkel, T.A., and L.A. Loeb (1981) Fidelity of mammalian DNA polymerases, Science, 213, 765-767. Lewis, P.N., W.J. Lukiw, U. DeBoni and D.R. Crapper-McLachlan (1981) Changes in chromatin structure associated with Alzheimer's disease, J. Neurochem., 37, 1193-1202. Mann, D.M.A., and P.O. Yates (1974) Motor neuron disease: the nature of the pathogenic mechanism, J. Neurol. Neurosurg. Psychiatry, 37, 1036-1046. M~7,arello, P., and M. Poloni (1988) Thiamine-monopho~phate diminution, idgntity of thiamine-pyrophosphatase to nucleoside-diphosphatase and possible pathogenetic implication for DNA tJamage in amyotrophic lateral sclerosis, in: T. Tsubaki and Y. Yase (Eds.), Amyotrophic Lateral Sclerosis, Elsevier, Amsterdam, pp. 319-324.

M ~ r e l l o , P., F. Focher, A. Verri and S. Spadari (1989) Mi~nc~rporation of uracil into DNA as possible contributor to neuronal aging and abiotrophy, Int. J. Neurosci., in press. Pedrali-Noy, G., and A. Weissbach (1977) HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase a, Biochim. Biophys. Acta, 477, 70-83. Seal, (3., and M.A. Sirover (1986) Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000 dalton catalytic subunit of DNA polymerase a, Proc. Natl. Acad. Sci. (U.S.A.), 83, 7608-7612. Sirover, M.A. (1979) Induction of the DNA repair enzyme uracil DNA-glycosylase in stimulated human lymphocytes, Cancer Res., 39, 2090-2095. So, A.G., and K.M. Downey (1988) Mammalian DNA polymerase a and 8: current status in DNA replication, Biochemistry, 27, 4591-4595. Spadari, S., F. Sala and (3. Pedrali-Noy (1982) Aphidicolin: a specific inhibitor of nuclear DNA replication in eukaryotes, Trends Biochem. Sci., 7, 29-32. Spadari, S., F. Focher and U. Hiibscher (1988) Developmental activity profile of DNA polymerases 8 and a in rat neurons suggests a coordinated in vivo function, In Vivo, 2, 317-320. Spadad, S., A. Montecucco, (3. Pedrali-Noy, G. Ciarrocchi, F. Focher and U. Htibscher (1989) A double-loop model for the replication of eukaryotic DNA, Mutation Res., 219, 147-156. Subba Rao, K., G.M. Martin and L.A. Loeb (1985) Fidelity of DNA polymerase-fl in neurons from young and very aged mice, J. Neurochem., 45, 1273-1278. Tye, B.-K., P.O. Nyman, I.R. Lehman, S. Hochhauser and B. Weiss (1977) Transient accumulation of Ok~7~kj fragments as a result of uracil incorporation into nascent DNA, Proc. Natl. Acad. Sci. (U.S.A.), 74, 154-157. Waser, J., U. Hiibscber, C.C. Kuenzle and S. Spadari (1979) DNA polymerase fl from brain neurons is a repair enzyme, Eur. J. Biochem., 97, 361-368. Weiss, B., and H.H. EI-Hajj (1986) The repair of uracil-containing DNA, in: M.G. Simic, L. Grossman and A.C. Upton (Eds.), Mechanism of DNA Damage and Repair, Plenum Press, New York, NY, pp. 349-356. Wilson, S., J. Abbotts and S. Widen (1988) Progress towards molecular biology of DNA polymerase fl, Biochim. Biophys. Acta, 949, 149-157.