Intracellular migration of DNA polymerase in early developing sea urchin embryos

5°

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96594

I N T R A C E L L U L A R MIGRATION OF DNA POLYMERASE IN E A R L Y DEVELOPING SEA URCHIN EMBRYOS L. A L O E B AND B F A N S L E R

The Institute/or Cancer Research Fox Chase, Phdadelphm, Pa. z 9 z z r (U S A ) (Received April 6th, 197 o)

SUMMARY

DNA polymerase has been purified from the nuclei of sea urchin embryos which were either grown in the presence of labeled leucine until hatching or exposed to labeled amino acids immediately prior to hatching. In both experiments the extent of labeling of the most purified fraction is approximately the same as that of the bulk protein. Evidence indicates there is little protein turnover during early development. Previously we have shown that there is a change in locahzation of DNA polymerase activity from the cytoplasm to the nucleus in early developing sea urchin embryos. The lack of preferential synthesis shown here is evidence that this change in locahzatlon represents a physical migration of the enzyme.

INTRODUCTION

During the first 16 h of exponential cell davlsion and rapid DNA synthesis after fertilization in S. purpuratus embryos, there is no significant change in the whole embryo DNA polymerase activity I. However, we have shown a marked change in the intracellular localization of polymerase activity. After each division cycle, progressively more activity is recovered in isolated nuclei while the corresponding cytoplasmic fractions show a concomitant loss of activity 1,2. We now consider the change in localization of DNA polymerase m relation to the extent of synthesis and turnover of the enzyme. Evidence indicates that during early development of sea urchin embryos only a fraction of the total protein undergoes turnover 3. We find polymerase is not an exception. It must have been synthesized during oogenesis and stored in the cytoplasm of the egg. During early development the enzyme migrates to the newly forming nuclei, thus maintaining a special relationship between nuclear DNA and polymerase.

MATERIALS AND METHODS

With the exception of those described below, all other procedures used in this investigation are identical with those previously reported 1,2. Sea urchin nuclear DNA polymerase was purified by the method of LOEB4 and the steps indicated here are consistent with that procedure. Chromatography on Bwchzm B w p h y s Acta, 217 (197 o) 50-55

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51

hydroxylapatite was omitted since it did not increase the purity of the enzyme. All steps are carried out in the presence of 20 ~o (w/v) glycerol or I M dextrose: the enzyme requires these or other polyglycols for stability4. DNA polymerase was assayed throughout the purification by using " a c t i v a t e d " calf thymus DNA as a primeI4. One unit of activity is defined as the amount of activity required to convert I re#mole of labeled deoxynucleoside triphosphate into acid-insoluble material in IO min at 37 °. Based on initial rate of incorporation, a unit of sea urchin nuclear polymerase is equivalent to about 1.2 units of Escherichia coli DNA polymerase 5 or 6.0 units of calf thymus DNA polymerase ~. Deoxyribonuclease activity was assayed with partially degraded sea urchin 8H-labeled DNA as a substrate4.The amount of labeled amino acid incorporated into protein was determined b y precipitating the protein with i M HCI04, reprecipitating from 0.2 M N a O H and dissolving in I ml of Hyamlne chloride (Packard Instrument Co.). Thereafter, a toluene-phosphor solution was added and radioactivity determined 4. A standard solution of E~H~toluene was added to each vial and results are given in disint./min.

RESULTS

In order to measure the extent to which DNA polymerase was synthesized during early development the enzyme was purified from hatched S. [ranciscanus embryos which had been grown in the presence of L-[SHlleucine (Table I, Expt. A). If polymerase was preferentially synthesized, relative leucine incorporation (all dlsint./min per mg protein) would increase as each fraction was selectively enriched for the enzyme. Even though the extent of polymerase purification was over 3oo-fold, the amount of [3H]leucine which had been incorporated into the most purified fraction (17 90o disint./min per mg protein) was not significantly different from that of the unfractionated embryonic proteins (19 500 disint./min per mg protein). Furthermore, there was little variation in extent of leucine labeling a m o n g the various fractions. These results indicate that during early development DNA polymerase does not undergo selective turnover. Of note is the increased leucine incorporation into certain proteins of the nuclear fraction; these do not copurify with the polymerase but are excluded during phase separation. Studies of NEMER AND LINDSAY~ indicate that these proteins m a y be histones which are preferentially synthesized during the 32-I28-cell period. The preceding experiment provides evidence of noppreferential turnover (synthesis and degradation) of polymerase during the first 16 h of development. We now ask, what is the rate of synthesis of the enzyme compared to the bulk protein at the time immediately prior to hatching? S. purpuratus embryos at this stage were exposed to ~H-labeled amino acids for 30 min and then harvested. DNA polymerase was purified from these embryos and the incorporation of 3H into the different protein fractions was determined (Table I, Expt. B). As a source of precursors for protein synthesis, an algal hydrolysate of labeled amino acids offered two distinct advantages: since m a n y amino acids were labeled, there was less selection for proteins which have an exceptionally high composition of a single amino acid. Second, a greater amount of radioactivity was incorporated for a given amount of protein synthesized. Amino acid incorporation in the most purified fraction is only a little greater than B*och*m. B*ophys. Acta, 217 (197 o) 5o-55

52

L. A. LOEB, B. FANSLER

TABLE I PURIFICATION

OF all-LABELED

SEA URCHIN

DNA

NUCLEAR

POLYMERASE

I n E x p t . A, o 5 mC of L-[SH]leuclne w a s a d d e d to a c u l t u r e 15 rain a f t e r f e r t i l i z a t i o n w h i c h cont a m e d 800 ml of e m b r y o s a n d t h e c u l t u r e w a s h a r v e s t e d a t h a t c h i n g (16 h). I n E x p t . B, 0. 5 mC of a n a l g a l h y d r o l y s a t e of [SH]amIno acids (Schwarz B I o R e s e a r c h ) w a s a d d e d t o a c u l t u r e cont a m i n g 2o0 ml of e m b r y o s 3 ° m m p r i o r to h a r v e s t i n g a t t h e h a t c h i n g stage.

Fract2on and step

Expt A (S. lranc*scanus)

I II III IV

Whole embryo Nuclei Phase separation (NH~)2SO 4 Acid p r e c i p i t a t i o n V Phosphocellulose VI D E A E - c e l l u l o s e VIII Sephadex

Expt. B (S. purpuratus)

Polymerase act*wry (umts/mg)

L-[3H]leuc*ne Polymerase *ncorporated aelzvzty (d~s*nt./m~n (umts/mg) per mg X Io -2)

[3H]Am*no aczds ,ncorporated (d~sznt./m,n per mg × zo -~)

o 88 2 84 7 60 12 2 12. 7 73 2 187 o 270.0

195 342 116 139 156 320 187 179

61" 76 67 119 49 53 iii iio

1.2o 7.45 lO.75 18.4 21.8 85 o 288 o 425 o

* ] n c o r p o r a t l o n of [3H]amlno acids i n t o w h o l e e m b r y o s w a s d e t e r m i n e d in t w o s e p a r a t e e x p e r i m e n t s . 3 m l of h a t c h e d e m b r y o s were e x p o s e d t o o I mC of t h e s a m e a l g a l h y d r o l y ~ a t e m i o o m l of sea w a t e r for 3 ° min. E m b r y o a n d n u c l e a r f r a c t i o n s were o b t a i n e d . The a v e r a g e incorp o r a t i o n i n t o w h o l e e m b r y o s a n d n u c l e i w a s 69' lO s a n d 86- Io s d i s m t . / m l n p e r m g prot e i n, res p e c t i v e l y . T h e i n c o r p o r a t i o n in t h e w h o l e e m b r y o f r a c t i o n w a s t a k e n t o be 8o % of t h e n u c l e a r i n c o r p o r a t i o n t h a t w a s o b t a i n e d in t h i s p u r i f i c a t i o n procedure.

that of the nuclei, indicating that during the purification there was no selection for a rapidly synthesized protein. The final step in the purification of the polymerase labeled with [3HJamino acids (Table I, Expt. B) is shown in Fig. I. A complete separation of deoxyribonuclease (endonuclease) activity and DNA polymerase activity was achieved. The spe-

_E

& 13

ml 2200=-

20

8_

C

"Em 3=

m o

Am,noAc,d~ - _ Polymerose Incorporofed f ~.

IIO0-

=*5

[

\-'NL Protein

Deoxyrlbonucleose

o

#_ E

__~oo 30

40

5og g ~

Frachon Number Fig. I. Gel f i l t r a t i o n of sea u r c h i n n u c l e a r D N A p o l y m e r a s e in S e p h a d e x G -i oo. 200 u n i t s of F r a c t i o n V I were a p p l i e d a n d e l u t e d from t h e c o l u m n 4.

Bzoch~m Bzophys Acta, 217 (197 o) 50-55

SEA URCHIN NUCLEAR D N A POLYMERASE

53

cific activity of the polymerase (units/mg protein) was nearly constant over the major part of the peak. Furthermore, the ratio of amino acid incorporation (all disint.] min) to #g protein did not significantly vary among the fractions of the peak.

DISCUSSION

Early development of sea urchin embryos is characterized by exponential cell division. The cells divide in 2 h or less, and the DNA, amounting to 1.8. IO-1~ g per diploid nucleus, can double in about IO-I2 rain at 15 ° (ref. 7). This high rate of DNA synthesis is accompanied by an exceptionally high level of DNA polymerase activity ~n vitro 8, the specific activity being greater than other reported eucaryotic tissues. We have shown previously that the total DNA polymerase activity per embryo remains nearly constant throughout the first 16 h of development 1. Further studies demonstrated that the great majority of the polymerase activity was found in the cytoplasm of the egg1. As the embryo proceeded through successive cell divisions, progressively more polymerase activity was found in the nuclear frachon with a concomitant loss of activity in the cytoplasm. By hatching, 2oo-4oo-cell stage, up to 95 ~/o of the polymerase activity was recovered in nuclei 1,z. The ratio of DNA polymerase activity to the DNA content in nuclei isolated at Intermediate stages before hatching was nearly constant, i.e. the ratio of activity to DNA was 35, 39, 31 and 25 umts per mg DNA in 2-, 8-, 32- and I28-cell embryos. This translocatlon of poly~ merase activity could have resulted from either a breakdown in the cytoplasm and preferential synthesis of polymerase in nuclei, extremely rapid turnover in cytoplasm with some transfer to nuclei, or a physical migration of a stored polymerase from the cytoplasm to the nucleus. To distinguish between these possibilities we have looked for evidence of selective synthesis of DNA polymerase. If there is a rapid breakdown and resynthesis of the enzyme, it is reasonable to assume that during resynthesis the enzyme would incorporate radioactive amino acids added to the culture. The embryos are readily permeable to exogenous amino acids and we find no evidence for special pools of precursors destined for the synthesis of particular proteins. If polymerase is selectively synthesized in embryos grown in the presence of labeled amino acids, the enzyme would contain proportionally more radioactivity than the bulk of the cell proteins. Upon purification the ratio of disint./mm per mg protein would increase with greater enrichment of the polymerase. The alternative to selective synthesis in the nucleus or cytoplasm during early development involves the migration of a preformed cytoplasmic enzyme. In this case, one would expect the enzyme to undergo little protein turnover at a rate no greater than the bulk embryo protein. During purification of the polymerase the ratio of radioactivity to protein would not increase This latter result was observed. The ratio of aH dlsint./mln per mg protein in the most purified polymerase fraction was not markedly different from that In the bulk of the nuclear protein. Evidence indicates that there is little protein turnover in early developing S. p u r p u r a t u s embryos 8. The amount of protein (4° #g) in the unfertilized egg is no different than that of the 4oo-cell embryo 8. Upon fertilization there is an immediate increase in amino acid incorporationg; thereafter the rate of protein turnover remains Bzoch,m. B~ophys. Acta, 217 (197 o) 50-55

54

L . A . LOEB, B. FANSLER

constant 3. During the first 16 h of development there is only 20 % turnover of the total protein from the unfertilized egg (calculated from leucine and valine incorporation data in ref. 3). Since polymerase is not selectively synthesized, no more than 20 % would undergo turnover. This is insufficient, m terms of breakdown and resynthesis, to account for the 90 % change in localization of polymerase activity from the cytoplasm to the nucleus. Therefore, the observed change in the localization of DNA polymerase activity reflects a physical migration of the enzyme. Polymerase must have been synthesized during oogenesis and stored in the cytoplasm of the egg. With successive replications after fertilization, the polymerase is quantitatively transferred to newly formed nuclei. Some attention must be given, at this point, to the composition of the most pure fraction (Fraction VIII) from the purification. As shown in Fig. I, this final step, Sephadex gel filtration, yields a single symmetrical retarded peak of protein comcident with polymerase activity. The 3H dasint./min indicative of amino acid incorporation give a remarkably unvarying ratio (SH dlsint./min per #g protein) an the fractions comprising the peak. In addition to evidence reported in this paper, chromatographic, chemical and electrophoretic studies indicate that the most purified fraction of the enzyme is homogeneous 4. Rechromatography of the most purified fraction by a variety of methods yielded a single symmetrical retarded peak. Chemical analysis indicated that if DNA was present, it is less than 0.5 %. Fraction VIII exhibited one distinct migrating protein band after electrophoresls in polyacrylamlde gel. We conclude that the major part, if not all, of the protein in the most purified fraction is DNA polymerase. The only other purified proteins which uniquely participate in cell division are those of the mitotic apparatus. Studies on the synthesis of these proteins during the first cell division of sea urchin embryos indicate that they are not synthesized at a rate exceeding that of the bulk cell proteins 1°. If these studies can be generalized, a molecular catalog of an egg would include most of the enzymatic and structural components necessary for rapid replication for a number of cell generations. It is possible that the intracellular migration of DNA polymerase is limited only to very early development. Alternatively, this migration may occur prior to DNA synthesis in all eucaryotlc cells, but the requirements for rapid DNA synthesis during early development in these embryos has given us the opportunity to detect it. The results with partially synchronized L-cells reported by LITTLEFIELD et al. n and GOLD AND HELLEINER12 support the latter concept; during DNA synthesis a decrease in the polymerase activity of the supernatant fraction and an increase in the particulate fraction were noted.

ACKNOWLEDGMENTS

This investigation was supported by grants from the American Cancer Society (E-483) and from the Stanley C. Dordick Foundation. Support was also derived from grants to this institute: U.S. Public Health Service grants CA-o6927 and FR-o5539, American Cancer Society grant IN-49, and an appropriation from the Commonwealth of Pennsylvania. We thank Drs. Darnel Mazia, Martin Nemer and Jack Schultz for generous counsel. Bzoch~m. B~ophys. Acla, 2i 7 (197o) 5o~55

SEA URCHIN NUCLEAR D N A

POLYMERASE

55

REFERENCES I 2 3 4 5 6 7 8 9 IO II 12

B. FANSLER AND L. A. LOEB, Exptl. Cell Res., 57 (1969) 305 • L. A. LOEB, ]3. FANSLER, R. WILLIAMS AND D. MAZlA, Exptl. Cell Res., 57 (1969) 298 B. J. FRY AND P. R. GROSS, Develop. Bzol., 21 (197 o) 125. L. A LOEB, [. Bwl. Chem., 244 (1969) 1672. C C. RICHARDSON, C. L. SCHILDKRAUT, H. V. APOSHIAN AND A KORNBERG, J BIol Chem, 239 (1964) 222. M. YONEDA AND F J BOLLUM, J. B,ol. Chem., 240 (1965) 3385 M. NEMER AND D T. LINDSAY, B*ochem. 13$ophys Res Commun., 35 (1969) 156. L A LOEB, D. MAZlA AND A. D. RUBY, Proc Natl. Acad. Se* U.S., 57 (1967) 841. D. EPEL, Proc. Natl. Acad Scz. U S., 57 (I967) 899 F H. WILT, H SAKAI AND n . MAZIA, J. Mol B~ol., a 7 (1967) i. J. W. LITTLEFIELD, A P. McGovERN AND K. B. MARGESON, Proe. Natl Mead Sc* U S , 49 (1963) lO2. M GOLD AND C W HELLEINER, Bzoch*m B',ophys Acta, 80 (1064) 193

B~ochzm Bzophys Acta, 217 (197 o) 5o-55