Planta (Bet1.) 127, 69--75 (1975) 9 by Springer-Verlag 1975

Development of Chromatin-bound and Soluble DNA Polymerase Activities during Germination of Pisum sativum L.* Nigel E. Robinson** and John A. Bryant *** Nottingham University School of Agriculture, Sutton Bonington, Loughborough, LE 12 5RD, U.K. Received 25 June; accepted 28 July 1975 Summary; Chromatin-bound and soluble DNA-dependent DNA polymerases were assayed in the embryonic axes of germinating peas (Pisum sativum L.). Activities of both enzymes increased markedly during germination, prior to the onset of net DNA synthesis. The activity of the soluble polymerase showed a better temporal correlation with net DI~A synthesis than did the activity of the chromatin-bound polymerase. The activity of the soluble polymerase greatly exceeded the activity of the chromatin-bound polymerase, particularly during the period of DNA replication.

Introduction Mammalian cells contain two major DNA-dependent DNA polymerases, one of which is tightly hound to the chromatin, and the other of which is freely soluble. (Chang and Bollum, 1972 ; Chang et al., 1973 ; Craig and Keir, 1975). The precise roles of the two enzymes in DNA replication have not been clarified, but the activity of the soluble enzyme is much better correlated with DNA replication than is the activity of the chromatin-bound enzyme (Chang and Bollum, 1972; Chang et al., 1973; Craig et al., 1975). Soluble and chromatin-bound DNA polymerases have been reported from a number of higher plants (Stout and Arens, 1970; Wever and Takats, 1970; Howell and Hecht, 1971; Mory etal., 1972; Dunham and Cherry, 1973). The evidence concerning the relationship of the two enzymes to DNA replication is fragmentary. In L i l i u m microspores, soluble DNA polymerase activity reaches a peak during the S-phase of the mitotic cycle, but the peak of activity is not very marked (Howell and Hecht, 1971). In Tradescantia pollen grains, there is no correlation between chromatin-bound polymerase activity and DNA synthesis (Takats and Wever, 1971). In excised beet tissue, chromatin-bound DNA polymerase activity increases prior to the onset of net DNA synthesis which follows excision of tissue slices from dormant storage organs (Dunham a n d Cherry, 1973). Similarly, excised Jerusalem artichoke tissue exhibits an increase in "total" DNA polymerase activity which precedes n e t DNA synthesis (Harland et al., 1973). In germinating wheat embryos , the onset of not DNA Synthesis is preceded by an eight- to ten-fold increase in the *Abbreviations: dATP~deoxyadenoSine 5'-triphosphate; dCTP=deoxycytidine 5'-triphosphate; dGTP-----deoXyguanosine5'-triphosphate; d T T P : deoxythymidine5'-triphosphate; TCA:trichloroacetic acid. ** Present address: Department of Botany, School of Biological Sciences, University of Leicester, Leicester, U.K. *** Present address: Department of Botany, University College, Cardiff CF1 1XL, U.K


N.E. Robinson and J. A. Bryant

a c t i v i t y of soluble D N A polymerase (Mory et al., 1972). There h a v e been n o reports of investigations i n which t h e c h r o m a t i c - b o u n d a n d t h e soluble D N A polymerase h a v e been s t u d i e d i n the same plant. I n the previous p a p e r (Robinson a n d B r y a n t , 1975) we reported t h a t n e t D N A synthesis i n t h e e m b r y o n i c axes of g e r m i n a t i n g peas is i n i t i a t e d at a p p r o x i m a t e l y 30 h after t h e onset of g e r m i n a t i o n . The g e r m i n a t i n g pea is therefore a useful system for s t u d y of the relationship b e t w e e n D N A polymerase a c t i v i t y a n d D N A replication, a n d also for s t u d y of t h e role of D N A polymerase in t h e control of t h e onset of D N A replication d u r i n g g e r m i n a t i o n . T h e results of such studies are r e p o r t e d i n this paper.

Materials and Methods Growth o/plants. Pea seeds (Pisum sativum, L., e. v. Feltham First) were surface sterilised in sodium hypochlorite solution (2.5 % w/v available chlorine) for 15 rain, washed for four hours in running tap water, planted in moist vermiculite and allowed to germinate at 22~ in the light. The beginning of the surface-sterilisationperiodwas takenastheonset of germination. At intervals after the onset of germination, samples of 40 seeds were removed. The embryonic axes were dissected out and used for assay of chromatin-bound and soluble D57A polymerases. D N A polymerase assays. The optimum conditions for assay of the enzymeswere determined in a series of preliminary experiments. The assay procedures described here were those that provided the optimum assay conditions. (i) Chromatin-bound DNA polymerase: Embryonic axes were homogenised at 4~ with a pestle and mortar, in two volumes of 50 mM Tris-HC1 buffer, pH 7.25, containing I mM magnesium acetate, 1 mM Na 2 EDTA, 12.5 m ~ sucrose and 10 mM mereaptoethanol. The homogenate was filtered through 30-35 tzm nylon mesh and then centrifuged at 2 000 • g for 30 rain at 1~ The precipitate was a crude preparation of chromatin, contaminated by fragments of cell wall. Further purification of the chromatin did not lead to any enhancement of DNA polymerase activity. The crude ehromatin preparation was re-suspended in 100 m ~ Tris-HC1 buffer, pH 7.25, containing 10 mM magnesium acetate and 8 mM mercapto-ethanol. Aliquots of 0.1 ml of the chromatin were used for each assay, in a total volume of 0.3 ml. The final concentrations in the 0.3 ml assay solution were as follows: 50 mM Tris-ttCl, pit 7.25, 5 m ~ magnesium acetate, 4 m2r mercapto-ethanol, dATP, dCTP and dGTP each at 1.3 mM, 0.7 mM dTTP, 5 IzCi [Me-aHJdTTP (15-30 Ci/mmol) and native calf-thymus DNA (330~g/ml). The DNA was not required as a template by the ehromatin-boundpolymerase, but was present in order to prevent the degradation by chromatin-bound deoxyribonuelease of the labelled DNA synthesised by the polymerase during the assay. Assay tubes were incubated at 37~ with shaking, for periods of zero to 30 rain. The reaction was stopped by the addition of one ml of ice-cold 5% TGA, containing 2 mM sodium pyrophosphate. Unpolymerised radioactive dTTP was removed by repeated cycles of dissolving the precipitates in 0.2 M NaOH followed by repreeipitation by 7% ttC10~, as described by Harland et al. (1973). Precipitates were finally collected on Whatman GF-G glass-fibre filters which had been presoaked in TCApyrophosphate. The filters were washed with 10 ml TCA-pyrophosphate followed by 10 mls ethanol, dried, and then counted in toluene-PPO-POPOP scintillation cocktail. The washing procedure described here routinely reduced the level of radioactivity in the zero-time assays to within 15 to 30 cpm of the background level. Absolute counting efficiencies were determined by addition of 0.1 ml aliquots of chromatin to different amounts of [aH]DNA of known specific activity. The mixtures were precipitated with TCA and then washed and counted as described above. (ii) Soluble DNA polymerase: Tissue was homogenised as described for the chromatinbound enzyme, except that the pI-I of the Tris-HC1 buffer was 8.1, and the magnesium acetate was at a concentration of 15 raM. The homogenate was filtered through 30-35 izm nylon mesh, and centrifuged at 34 000 X 9 for 30 rain at 1~C. The supernatant was used as the enzyme solution. Aliquots of 0.1 ml of supernatant were used for each assay in a total volume of 0.3 ml. The constituents of the 0.3 ml of assay solution were as described above, except that the pH

DNA Polymerase Activities during Germination


was maintained at 8.1, and the magnesium acetate was at 15 mlVLThe remainder of the assay procedure and determination of counting efficiencies were carried out as described for the chromatin-bound enzyme. Results

and Discussion

Properties o/the two DNA


I n these experiments, two different types of DNA polymerase were detected. One sedimented at 2000 • g, and is clearly equivalent to the chromatin-bound D N A polymerase described in mammalian cells. The other was not sedimented by centrifugation at 34000 • g, and is thus equivalent to the soluble or "cytoplasmic" DNA polymerase of mammalian cells. The chromatin-bound enzyme from peas exhibited a p t t optimum of 7.25 and magnesium concentration optimum of 5 mM. The soluble polymerase showed a p H optimum of 8.1 and a magnesium concentration optimum of 15 mM. These properties are compared with those of DNA poIymerases from other plants in Table 1. Under optimum assay conditions at 37~ both the ehromatin-bound polymerase and the soluble polymerase exhibited linearity for only ten rain (Fig. 1.), although under sub-optimal conditions, linearity often extended to 30 min. The reason for the marked decline in rate after ten rain is not clear, but it cannot be ascribed to exhaustion of the substrate supply. I t is possible t h a t the short-term linearity is only a feature of assays carried out with crude extracts, since both the chromatin-bound and the soluble polymerases exhibit linearity for at least 45 min after purification b y DEAE-cellulose chromatography (C. Stevens and J . A . Bryant, manuscript in preparation). Both enzymes, and particularly the soluble enzyme, exhibited significant activity when d G T P was omitted from the assay solution (Table 2). The ability to sustain a significant level of deoxyribonucleotide polymerisation in the absence of one of the deoxyribonueleoside triphosphates is a feature exhibited by a number of other DNA polymerases from plants (Wever and Takats, 1970; Stout and Arens, 1970.) and by some mammalian DNA polymerases (Craig and Keir, 1975). The soluble enzyme was almost completely dependent on added DNA (Table 2). We were not able to demonstrate DNA dependence for the ehromatin-bound polymerase, since chromatin contains DNA. However, it has recently been established t h a t the chromatin-bound enzyme does exhibit DNA dependence after it has been solubilized and partially purified (C. Stevens and J . A . Bryant, manuscript in preparation). For both the ehromatin-bound and soluble enzymes, the product of

Table 1. Magnesium concentration and pH optima of DNA polymerase from plants Species

P i s u m sativum Beta vulgaris Tradescantia paludosa T r i t i c u m vulgare Zea mays

Chromatin-bound Soluble






7.25 8.0 7.6

5 talk 5 mM 5 mM




15 m_NI --4 mM 5 mM


-7.6 8.4

This paper Dunham and Cherry (1973) Wever and Takats (1970) Mory et al. (1972) Stout and Arens (1970)


N.E. Robinson and J. A. Bryant





._~ 50


y i













, 20




j 40


Fig. la and b. Linearity of DNA polymerase assays under optimum conditions at 37~ (a) Chromatin-bound polymerase. (b) Soluble polymerase Table 2. Effect of composition of reaction mixture on DNA polymerase activty Reaction mixture

Relative enzyme activity Chromatin-bound Soluble

complete minus Mg~+ minus dGTP minus DNA plus DNase a "native" calf.thymus DNA replaced by: "nicked" calf-thymus DNA denatured calf-thymus DNA native pea DNA

100 7.5 21.8 -0.0

100 12.5 58.0 3.9 8.0


102 70 14

a Reaction mixture treated with deoxyribonuclease after stopping the reaction. the reaction was degraded by deoxyribonuclease (Table 2). The properties exhibited by the ehromatin-bound and soluble enzymes in peas thus indicate that they are both DNA-dependent DNA polymerases. I n the experiments described here, "native" calf-thymus DNA was routinely used as a template ~or the soluble DNA polymerase. The effieiencies of templates other than "native" calf-thymus DNA are shown in Table 2. "Nicked" calfthymus DNA (calf-thymus DNA into which single-stranded breaks had been introduced by the method of Aposhian and Kornberg, 1962) was utilised to the same extent as "native" calf-thymus DNA. Heat-denatured DNA (i.e. single-stranded DNA) was much less efficiently used as a template. The high level of activity sustained by the enzyme with "native" calf-thymus DNA as a template is surprising. I t seems very likely that the particular batch of commercially:available calfthymus DNA Used in these experiments was in fact "nicked". I n more recent experiments, using a different batch of commercial calf-thymus DNA, the efficiency of native calf-thymus DNA as a/template was much less than that of "nicked" DNA, and was similar to that of native pea DNA.

DNA Polymerase Activities during Germination



0.15 h


o~ 0.05 /

/ -5 E c








f I


40 Hours of germinotion

Fig. 2a and b. Development of DNA polymerase activity in the embryo axis during germination. (a) Chromatin-bound polymerase. (b) Soluble polymerase

D N A Polymerase Activity during Germination The patterns of development of the activities of the two polymerases are shown in Fig. 2. These patterns were obtained reproducibly in a number of experiments, and the data shown are taken from one representative experiment in each ease. Both the ehromatin-bound polymerase and the soluble polymerase were detectable as early as 6 h after the onset of germination, and it is likely t h a t both enzymes are present in dry seeds prior to the onset of germination. The activity of the chromatin-bound enzyme increased three-fold between six and 22 h, after which there was no further increase in activity. The solubIe enzyme showed no increase in activity between six and 14 h, but between i4 and 30 h, the activity increased 4-fold. There w a s little further increase between 30 and 44 h. At all times, the activity of t h e soluble enzyme was much greater t h a n t h a t of the chromatin-bound enzyme. This was particularly true of the period following the onset of net D N A synthesis, i.e. from 30 h, onwards (Robinson and Bryant, 1975), when the soluble polymerase was 80 to 100 times as active as the ehromatin-bound polymerase. I n order to study the relationship of the activities of t h e DNA polymerases to DNA replication, it is necessary to compare the activities with the observed rates of DNA accumulation. From the measured rates of increase of the DNA content of the embryonic axis (Robinson and Bryant, 1975) it is possible to calculate the rate of dTMP incorporation achieved b y the embryonic axis in vivo (Table 3). I n making these calculations, we h a v e assumed t h a t no DNA turnover occurs. Since it is likely t h a t there was in fact some DNA turnover (Bryant et al., 1974; Robinson and Bryant, 1975), the actual rates of dTMP incorporation in vivowere likely to have been slightly greater t h a n our calculated rates. The activities of the two polymerases were routinely assayed at 37~ whereas the pea seeds were grown at 22~ However, during the determination of the temperature optima of the enzymes, a number of assays were carried out at 22~ We have therefore been able to convert activities at 37~ to activities at 22~ thereby enabling us to compare directly the activities of the polymerasos with the


, N. E. Robinson and J. A. Bryant Table 3. Comparison of DNA polymerase activities with rates of DNA replication Age of embryonic axis (h)

6 14 22 30 44

nmol dTMP/embryoaxis/h at 22~ C Rate of net DNA synthesis

Chromatin- Soluble bound polymerase polymerase

0 0 0 0.82 0.82

0.30 0.59 0.86 0.68 0.88

16.6 18.4 40.6 68.6 72.8

rates of DNA replication (Table 3). I t is very clear t h a t the assayed activity of the chromatin-bound D N A polymerase was only just able to account for the observed rate of DNA replication, whereas the activity of the soluble polymerase was greatly in excess of t h a t required to sustain D N A replication. This holds true even when the much lower activities achieved with native pea DNA as a template are considered (see Table 2). Further, the attainment of maximal activity of the ehromatin-bound enzyme occurred several hours before the onset of net DIqA synthesis, whereas the maximal activities of the soluble enzyme coinCided closely with the onset of DNA replication. The relationship between the activities of the two enzymes and the replication of DNA in the embryonic axis of the pea is therefore very similar to t h a t observed in mammalian cells (see Introduction). Although these data are consistent with the view t h a t the soluble polymerase is involved in D N A replication, whereas the chromatin-bound polymerase is involved in D N A repair or turnover, there is as yet no direct evidence for this view. We are currently investigating the properties of the two enzymes, and their occurrence in dividing and non-dividing cells, in order to gain insights into their roles i n vivo. From the data presented here, it is probable t h a t the increase in the activity of soluble DNA polymerase is a prerequisite for the onset of DNA replication during germination, as has been demonstrated in germinating wheat embryos (5~ory et al., 1972). However, it is unlikely t h a t the increased DNA polymerase activity is the only factor contributing to the control of DNA replication, particularly since the activity of the soluble polymerase is far in excess of t h a t required to sustain D N A replication, even in the period prior to the increase in activity. I t is thus likely t h a t other factors act to control D N A replication during germination. The nature of these factors is currently under investigation. N.E.R. thanks the Ministry of Agriculture, Fisheries and Food for a post-graduate scholarship. References

Aposhian, H.V., Kornberg, A.: Enzymatic synthesis of deoxyribonucleic acid. IX" The polymerase formed after T 2 bacteriophage infection of Escherichia coli: a new enzyme. J. biol. Chem. 237, 519-525 (1962)

DNA Polymerase Activities during Germination


Bryant, J . A . , Wildon, D.C., Wong, D.: Metabolically labile DNA in aseptically grown seedlings of Pisum sativum L. Planta (Berl.) 118, 17-24 (1974) Chang, L. M. S., Bollum, F. J. : Variation of deoxyribonucleic acid polymeraso activities during rat liver regeneration. J. biol. Chem. 247, 7948-7950 (1972) Chang, L. M. S., Brown, M., Bollum, F. J. : Induction of DNA polymerase in mouse L cells. J. melee. Biol. 74, 1-8 (1973) Craig, R. K., Costello, P. A., Keir, H. 1~I.: Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Relationship to the physiological state of the cells, and to synchronous induction of synthesis of deoxyribonucleic acid. Biochem. J. 145,233-240 (1975) Craig, R. K., Keir, It. M.: Deoxyribonucleic acid polymerases of BtlK-21/C13 cells. Partial purification and characterization of the enzymes. Biochem. J. 145, 215-224 (1975) Dunham, V. L., Cherry, J. It.: Multiple DNA polymerase activity solubflized from higher plant chromatin. Biochem. biophys. Res. Commun. 54, 403-410 (1973) Harland, J., Jackson, J. F., Yeoman, M. M. : Changes in some enzymes involved in DNA biosynthesis following induction of division in cultured plant cells. J. Cell SoL 18, 121-138 (1973) Howell, S. H., and Hecht, N. B. : The appearance of polynucleotide ligase and DNA polymerase during the synchronous mitotic cycle in JLilium microspores. Biochim. biophys. Acta (Amst.) 240, 343-352 (1971) Mory, Y. Y., Chen, D., Sarid, S.: Onset of deoxyribonucleic acid synthesis in germinating wheat embryos. Plant Physiol. 49, 20-23 (1972) Robinson, N. E., Bryant, J. A. : Onset of nucleic acid synthesis during germination of Pisum sativum L. Planta (Berl.) (preceding paper) Stout, E. R., Arens, M. Q. : DNA polymerase from maize seedlings. Bioehim. biophys. Aeta (Amst.) 218, 90-100 (1970) Takats, S. T., Wever, G. H. : DNA polymerase and DNA nuclease activities in S-competent and S- incompetent nuclei from Tradescantia pollen grains. Exp. Cell Res. 69, 25-28 (1971) Wever, G., Takats, S. T.: DNA polymerase activities in Tradescantia paludosa pollen grain nuclei. Biochim. biophys. Acta (Amst.) 199, 8-17 (1970)

Development of chromatin-bound and soluble DNA polymerase activities during germination of Pisum sativum L.

Chromatin-bound and soluble DNA-dependent DNA polymerases were assayed in the embryonic axes of germinating peas (Pisum sativum L.). Activities of bot...
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