Separation and characterization of protein factor of DNA polymerase B from developing rat brain

Biochimica et Biophysica Acta, 331 (1973) 54-60 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97850

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

JEN-FU CHIU* and S. C. SUNG

Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver 8 (Canada) (Received April 24th, 1973) (Revised manuscript received August 22nd, 1973)

SUMMARY

D N A polymerase B extracted from developing rat brain and subjected to (NH4)zSO4-fractionation and column chromatography on DEAE-cellulose lost its activity after Sephadex G-200 gel filtration due to loss of an activating factor. The partially inactivated D N A polymerase was markedly stimulated by the addition of this factor. The ability of the activating factor to enhance D N A polymerase B was destroyed by its treatment with a proteolytic enzyme as well as by heating. The effect of the activating factor on polymerase B does not appear to be a consequence of either nuclease or "nickase" activity.

INTROD UCTION

Mammalian cells contain multiple forms of D N A polymerases, which are distinguishable by their molecular weight and template requirements 1-6. Previous studies carried out in this laboratory have demonstrated that two D N A polymerases, A and B, are present in developing rat brain 5. The two D N A polymerases differ in their response to divalent cations, dithiothreitol, polyamines and different DNA templates 5'7. Enzyme A has a higher molecular weight than enzyme B 8. Our recent finding suggests that the induction of proliferative activity is primarily associated with D N A polymerase A in the developing rat brain 7. However, D N A polymerase B of the developing rat brain is more active than enzyme A in the cerebellum and cerebral cortex from older animals, and it is also present as particulate form in adult animals 7'9. The biological function of enzyme B is still not known. This communication describes results of experiments indicating the presence of an activating protein factor in D N A polymerase B of rat brain and its separation there from. Some properties of this protein factor are also described. * Present address: Department of Biochemistry, University of Texas at Houston, M. D. Ander son Hospital and Tumor Institute, Houston, Texas 77025 (U.S.A.).

PROTEIN FACTOR OF DNA POLYMERASE B

55

MATERIALS AND METHODS Materials

[3H]dTTP (15.7 Ci/mmole) was purchased from New England Nuclear Corporation. Unlabelled deoxynucleoside triphosphates, Cleland's reagent (dithiothreitol), calf thymus DNA and bovine serum albumin were obtained from Calbiochem. and pancreatic deoxyribonuclease was from Worthington Biochemical Corporation. [14C]DNA was prepared from Ehrlich ascites carcinoma cells ~°. Methods

DNA polymerase A and B from Wistar rat brain were prepared, as described in our previous paper 5, by extraction, (NH4)2SO4-fractionation and DEAE-cellulose chromatography. The "nickase" activity of the protein factor was assayed by the method of Baril et al. 11 with some modification. The reaction mixture contained the following in a final volume of 0.4 ml: 20/zmoles Tris-HCl buffer (pH 7.4), 2 #moles MgC12, 2 #moles dithiothreitol, 50 #g [14C]DN A from ascites tumor cells (0.1 mCi/g DNA) and an appropriate amount of the protein factor. After various incubation periods (I, 2 and 4 h), the reaction mixture was layered on a gradient of 5-20 ~o sucrose solution containing 0.3 M NaOH, 0.01 M EDTA and 1.0 M NaC1 and was centrifuged at 36 000 rev./min for 16 h at 0 °C in the SW 39 L rotor of the Spinco model L ultracentrifuge. After centrifugation, the bottom of the tube was punctured, 15-drop fractions were collected, and each fraction was measured for radioactivity. Deoxyribonuclease activity was measured as described by Sung 12 and protein concentration was determined by the method of Lowry et al. 13 with bovine serum albumin as a standard. RESULTS Separation o f D N A polymerase B into partially inactive enzyme and activator

In order to study the mechanism of enzymatic regulation of DNA polymerase B as well as its function, further purification of the enzyme by column chromatography with Sephadex G-200, phosphocellulose or hydroxylapatite was carried out. However, an unexplained loss of enzymatic activity was often encountered during the course of these procedures. As shown in Fig. 1, DNA polymerase B from 10-day-old rat brain was reduced markedly after passing through the Sephadex G-200 column (dotted line in Fig. 1). However, the activity of the enzyme was enhanced in the combination of Fraction 10 and 12 (dash line in Fig. 1), indicating the presence of an activating factor in Fraction 12. This factor was dissociated from the DNA polymerase during the course of purification. Nature o f the activating factor

The activity of DNA polymerase B was then assayed with or without the addition of the activating factor or with the factor treated as shown in Table I. The untreated factor increased the activity of the enzyme, but after treatment with trypsin it had lost its stimulatory capacity. The factor retained its activity when pretreated with trypsin together with trypsin inhibitor or when trypsin together with trypsin

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Fig. 1. Column chromatography of DNA polymerase B on Sephadex G-200. Enzyme B of DEAEcellulose fraction (3.5 mg of protein) was applied to a column of Sephadex G-200 (0.8 cm × 54 cm; equilibrated with 0.01 M phosphate buffer, pH 7.4 containing 2 mM fl-mercaptoethanol and 5 glycerol) and was eluted with the same buffer. Fractions of 3 ml each were collected at a flow rate of about 3 ml/h. Each tube was measured for absorbance at 280 nm and then assayed for DNA polymerase activity. - - , absorbance at 280 n m ; . . . , partially inactivated DNA polymerase activity; - - -, activated DNA polymerase activity. Fig. 2. Alkaline sucrose gradient centrifugation profile of native [~4C]DNA after incubation for 0, 1, 2 and 4 h in the presence of the protein factor. All procedures are as described in Methods. 0 , zero time incubation for control; A, 1-h incubation; A, 2-h incubation; (3, 4-h incubation. TABLE I N A T U R E OF THE ACTIVATOR OF DNA POLYMERASE B The activity of DNA polymerase B obtained from the Sephadex G-200 column was assayed with the activator or treated activator. Treated activator was obtained by preincubation of activator with 25 Fg trypsin or 25 Fg trypsin and 50 #g trypsin inhibitor at 37 °C for 15 min. After preincubation, 50/zg of enzyme B obtained from the Sephadex G-200 column was added to the reaction mixture and incubated at 37 °C for another 30 min.

Treatment

Activity (epm)

None +Activator +Activator (pretreated with trypsin then inhibitor added) +Activator (pretreated with trypsin and trypsin inhibitor) -I-Activator+trypsin+trypsin inhibitor +Activator (heated at 100 °C for 10 rain)

154 662 218 604 631 165

i n h i b i t o r w e r e a d d e d t o t h e i n c u b a t i o n m i x t u r e . T h e f a c t o r l o s t its a c t i v i t y w h e n h e a t e d a t 100 ° C f o r 10 m i n .

Nuclease activity of the factor T h e p o s s i b i l i t y o f t h e f a c t o r b e i n g n u c l e a s e w a s e x a m i n e d . 60/~g o f t h e p r o t e i n f a c t o r w a s first i n c u b a t e d w i t h [ 1 4 C ] D N A a t 37 ° C f o r 1 h a n d t h e n t h e r a d i o a c t i v i t y

PROTEIN FACTOR OF DNA POLYMERASE B

57

of the trichloroacetic acid precipitate and supernatant was measured. It is obvious from the results shown in Table II that the factor has no nuclease activity. However, it was possible that the factor might be a specific endonuclease or a "nickase", an enzyme which makes nicks on one of the D N A strands. To study this possibility the experiment was performed as described in the Methods. The results of centrifugation, shown in Fig. 2, suggest that the factor is not a "nickase" although a one-fraction shift of the peak tube was observed after 2 and 4 h incubation. The one-tube shift from the control experiment may be due to contamination by a trace amount of endonuclease or exonuclease. However, if the protein factor were a "nickase", it should have shifted the D N A peak closer to the top of the gradient 11 TABLE II NUCLEASE ACTIVITY OF THE FACTOR After the factor or pancreatic deoxyribonuclease was incubated with 20/~mole Tris-HCl buffer (pH 7.4), 0.15 mg ascites [~4C]DNA, 2/~mole MgCI2 and 2 #mole dithiothreitol at 37 °C for 1 h, 10 ~ trichloroacetic acid was added and the mixturewas centrifuged. The radioactivity of supernatant and precipitate was measured.

Sample

Radioactivity (cpm) Supernatant Precipitate

Factor (60/tg)

34 56 9050 8967 8978 8035

5/~g deoxyribonuclease 2/tg deoxyribonuclease 1/~g deoxyribonuclease

9234 9163 474 436 440 1295

The factor was then preincubated with D N A for 0, 7 and 15 min, respectively, before assaying the D N A polymerase activity of enzyme B. However, there was no difference in activity with various times of preincubation. The fact that the activities were independent of the length of preincubation time indicates that the activation is not an enzymatic process. This further suggests that the stimulation brought about by the activating factor was not due to "nickase" or endonuclease activity. If the factor were in fact a "nickase" or nuclease it would be anticipated that different times of preincubation would result in various degrees of breakage of the template and consequently in different amounts of template activity in the D N A polymerase assay.

Time course of stimulation The addition of 25/~g of protein factor to a reaction mixture containing 50/~g protein of enzyme B (Sephadex fraction) caused a marked increase in the rate of [3H]dTTP incorporation which remained constant throughout the course of incubation (Fig. 3). No detectable lag period was observed after the addition of the factor. The effect o f the activating factor on DNA polymerase A and B with various DNA as template As shown in Table III the activity of enzyme A was not stimulated by the

58

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Fig. 3. Kinetic studies on stimulation of DNA polymerase B by the factor. 25/~g of factor was added to the reaction mixture at 10 and 20 rain, respectively, after incubation commenced. Arrows indicate the time of addition of factor. ©, without factor; A, with factor. TABLE IIl EFFECT OF THE FACTOR ON DNA POLYMERASE A AND B WITH VARIOUS DNAs AS TEMPLATE The activities of enzymes A and B were assayed as mentioned in Materials and Methods, using various DNAs as template, in the absence (1) or presence (2) of the addition of the factor in the reaction mixture.

Enzyme

A (cpm) B (cpm)

DNA template Calfthymus (1) (2)

Sahnonsperm (1) (2)

Microcoecuslysodeikticus (1) (2)

263 556

375 411

191 286

227 980

415 1014

125 1237

activating factor, irrespective o f the source o f D N A used as template. However, the activity o f D N A p o l y m e r a s e B was e n h a n c e d by the activating factor with each o f the D N A templates tested. Hence the activating f a c t o r w o u l d a p p e a r to be an a c t i v a t o r specific for D N A p o l y m e r a s e B, b u t n o t for enzyme A. The e n h a n c e m e n t o f D N A p o l y m e r a s e B activity by the activating factor was greater with a D N A t e m p l a t e which h a d a higher ( G + C ) c o n t e n t as the template.

Effect of spermidine on DNA polymerase with protein factor W e have shown previously that s p e r m i d i n e greatly increases the activity o f D N A p o l y m e r a s e B b u t has no effect on the activity o f D N A p o l y m e r a s e A x4. T h e results in Table IV show t h a t the p a r t i a l l y inactivated p o l y m e r a s e can be stimulated s o m e w h a t b y either s p e r m i d i n e or by the p r o t e i n factor. However, the D N A p o l y m e r ase activity o b t a i n e d with b o t h spermidine a n d the activating factor present was considerably higher t h a n the sum o f the activities observed with either one alone.

PROTEIN FACTOR OF DNA POLYMERASE B

59

TABLE IV SPERMIDINE EFFECT ON DNA POLYMERASE A AND B WITH PROTEIN FACTOR Enzyme A was obtained from DEAE-cellulose chromatography 5 and enzyme B and the factor were then from Sephadex G-200 filtration of DNA polymerase B of DEAE-cellulose fraction 5. No spermidine

Enzyme Enzyme Factor Enzyme Enzyme

A B A + factor B ÷ factor

(cpm)

With spermidine (cpm)

201 127 26 200 300

194 238 22 191 1090

DISCUSSION

We have presented evidence to show that a protein factor is present in DNA polymerase B preparations which is removed from the enzyme by Sephadex G-200 gel filtration. The sensitivity of the activating factor to trypsin and to destruction by heat indicates that it is protein in nature. Moreover, the protein factor did not appear to possess nuclease or "nickase" activity. Thompson and McCarthy 15 found a cytoplasmic stimulator in ascites cell and L-cell cytoplasm. This stimulator differs from the activating factor described here since the former is heat stable. Shimada and Terayama 17 reported a factor in infant rat brain cytosol that stimulated DNA synthesis. This factor is thermostable and has a low molecular weight. The properties of activating factor described here are very similar to those of the "wedge" described by Erhan et aL 16. The wedge, a low molecular weight factor obtained from ascites fluid, stimulates DNA polymerase activity with double-stranded DNA as template, but has no effect on DNA synthesis with single-stranded DNA as template. The wedge is presumed to be an "unwindase". However the activating factor isolated from rat brain, described in this paper cannot stimulate the activity of DNA polymerase A even with native, double-stranded DNA as template and may differ from the wedge in this respect (Table IV). The elucidation of the mechanism of action of the protein activating factor, specific for DNA polymerase B, will be of considerable interest. ACKNOWLEDGEMENTS

This work was supported by a grant from the Medical Research Council of Canada.

REFERENCES 1 Chiu, J.-F. and Sung, S. C. (1970) Biochim. Biophys. Acta 209, 34-42 2 Wallace, P. G., Hewish, D. R., Venning, M. M. and Burgoyne, L. A. (1971) Biochem. J. 125, 47-54 3 Weissbach, A., Schlabach, A., Friedlender, B. and Bolden, A. (1971) Nat. New Biol. 231,167-170

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Chang, L. M. S. and Bollum, F. J. (1971) J. Biol. Chem. 246, 5835-5837 Chiu, J.-F. and Sung, S. C. (1971) Biochim. Biophys. Acta 246, 44-50 Baril, E. F., Brown, O. E., Jenkins, M. D. and Laszlo, J. (1971) Biochemistry 10, 1981-1992 Chiu, J.-F. and Sung, S. C. (1972) Nat. New Biol. 239, 176-178 Chiu, J.-F. and Sung, S. C. (1972) Biochim. Biophys. Acta 269, 364-369 Chiu, J.-F. and Sung, S. C. (1972) Biochem. Biophys. Res. Commun. 46, 1830-1836 Sung, S. C. and Quastel, J. H. (1963) Nature 200, 781-782 Baril, E., Brown, O. and Laszlo, J. (1971) Biochem. Biophys. Res. Commun. 43, 754-759 Sung, S. C. (1968) J. Neurochem. 15, 477-481. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265275 Chiu, J.-F. and Sung, S. C. (1972) Biochim. Biophys. Acta 281,535-542 Thompson, L. R. and McCarthy, B. J. (1968) Biochem. Biophys. Res. Commun. 30, 166-1 72 Erhan, S., Reisher, S., Franko, E. A., Kamath, S. A. and Rutman, R. J. (1970) Nature 2 2 5 , 3 4 0 342 Shimada, H. and Terayama, H. (1972) Biochim. Biophys. Acta 287, 415-426

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