Planta
Planta 143, 113-120(i978)
9 by Springer-Verlag 1978
Chromatin-bound DNA Polymerase from Higher Plants A DNA Polymerase-fl-like Enzyme
Christine Stevens*, J o h n A. Bryant **, and P. Carol Wyvill Department of Botany, UniversityCollege, P.O. Box 78, Cardiff CF1 1XL, U.K.
Abstract. Chromatin-bound DNA polymerase has been extracted from pea ( P i s u m s a t i v u m L.) seedlings, and partially purified by solubilization from chromatin followed by chromatography on columns of either DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAE-cellulose as a single peak, but is fractionated into two peaks, CI and CII, by DEAESephadex chromatography. If the enzyme is stored at - 1 5 ~ C for several days prior to chromatography, a third peak, CIII, derived from CII, is obtained. The polymerase is devoid of nuclease activity, and is relatively insensitive to N-ethyl-maleimide. These features, taken with the ion requirements and with data obtained from other plant species, lead to the suggestion that the chromatin-bound D N A polymerase of higher plants is similar to the D N A polymerasefi from vertebrates. Key words: Chromatin - D N A polymerase -
Pisum.
Introduction
Nearly all organisms so far investigated possess multiple DNA polymerases (EC 2.7.7.7.). Amongst the eukaryotes, the most widely studied are vertebrates, particularly mammals. Other than the mitochondrial D N A polymerase, three types of DNA-dependent D N A polymerase have been described in vertebrates (see reviews by Brun and Chapeville, 1977; Keir et al., 1977; McLennan and Keir, 1977). The major activity in cells undergoing D N A replication is DNA polymerase-c~. This is a soluble, high-molecularweight (1-2 • 105 daltons) enzyme which is sensitive * Present address ." TechnologyPolicyUnit, Universityof Aston, Gosta Green, BirminghamB4 7ET, U.K. ** To whom reprint requests should be addressed
to SH-group alkylating agents, such as N-ethyl maleimide. D N A polymerase-~ of vertebrates does not possess nuclease activity. The second type of polymerase is known as DNA polymerase-/L It is a lowmolecular-weight (ca. 5 • 104 daltons) enzyme, the majority of which is bound to the chromatin. It is insensitive to N-ethyl maleimide, and does not possess nuclease activity; its activity shows very little correlation with the rate of DNA synthesis. The third type of polymerase is D N A polymerase-7, which is distinguished mainly on the basis of its unusual ability to copy the polyribonucleotide strand of heteroduplexes such as A,.dT15. By contrast, lower eukaryotes, including algae, fungi and protozoans do not possess the low-molecular-weight D N A polymerase-/~ (see review by McLennan and Keir, 1977). Neither has D N A polymerase- 7 been unequivocally demonstrated in lower eukaryotes. Thus, all the D N A polymerases isolated from lower eukaryotes are of the DNA polymerase-c~ type, being soluble, high-molecular-weight enzymes which are sensitive to N-ethyl maleimide. Many lower eukaryotes possess two or even three such polymerases, and in these organisms, the activity of one of the polymerases is much better correlated with D N A replication than is the activity of the other(s) (Keir et al., 1977). Many of the D N A polymerases from lower eukaryotes differ from their counterparts in vertebrates in that they possess nuclease activity (McLennan and Keir, 1977; Ross and Harris, 1978). McLennan and Keir (1977) have proposed that during the course of evolution, the low-molecularweight chromatin-bound D N A polymerase-fl has arisen from an enzyme of the a-type by loss of nuclease activity accompanied by a decrease in molecular weight and further, that this change is associated with genome complexity. The higher plants are clearly crucial to this hypothesis, since in organisational terms, they are much less complex than vertebrates, 0032-0935/78/0143/0113/$ 01.60
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C. Stevens et al. : C h r o m a t i n - b o u n d D N A Polymerase from Higher Plants
but their genomes are at least as complex as those of vertebrates. Higher plants are certainly known to possess soluble D N A polymerases of the c~-type (Stout and Arens, 1970; Srivastava and Grace, 1974; Robinson and Bryant, 1975; Mory et al., 1975; Castroviejo et al., 1975; Gardner and Kado, 1976; Tymonko and Dunham, 1977; Stevens and Bryant, 1978). In no species of higher plant is there any clear evidence for two completely distinct e-type DNA polymerases, such as exist in lower euraryotes. The situation regarding the existence of DNA polymerase-fi in higher plants is unclear. Chang (1976) and Gardner and Kado (1976), using insensitivity to N-ethyl maleimide and low molecular weight as criteria for the identification of polymerase-/?, have claimed that higher plants do not possess this form of DNA polymerase. However, chromatin-bound DNA polymerase has been detected in Tradescantia (Wever and Takats, 1970), soybean (Leffler et al., 1971), tobacco (Srivastava and Grace, 1974), pea (Robinson and Bryant, 1975) and beet (Tymonko and Dunham, 1977). In this paper, we report a characterization of the chromatin-bound DNA polymerase from pea seedlings. We suggest that our data, taken with those obtained from other plants, particularly beet and tobacco, indicate that the chromatin-bound DNA polymerase of higher plants is a polymerase of the /3-type. Preliminary reports of part of this work have been published as abstracts (Stevens et al., 1975; Stevens and Bryant, 1976).
Materials and Methods
5mmolI-~ mercapto-ethanoI, 5 m m o l I - 1 Mg acetate and 1.5 m o l l i (NH4)2SO 4 (previously adjusted to p H 7.25 with N H 4 O H ). The suspension was stirred vigorously for two h at 2M ~ C, and then centrifuged at 34,000 x g for one h at 2 4 ~ C. The supernatant was de-salted by passage through a column (50 m m x 25 m m ) of Sephadex G-50 (fine), previously equilibrated with Tris-HCl-glycerol buffer, ph 7.25 (50 mmol 1- ~ Tris-HC1, 5 m m o l 1- ~ mercapto-ethanol, 1 m m o t l- ~ Na 2 E D T A and 25%, v/v, glycerol). The de-salted enzyme preparation was then applied to a column (160mm• of either DEAE-cellulose (DE 52) or DEAE-Sephadex (A-25). Protein was eluted from the column first with Tris-HCl-glycerol buffer, and then a linear gradient of 0.0 to 1.0 mol 1- a KCI in l'ris-HCl-glycerol buffer. Fractions of 2.7 ml were collected automatically, monitored for absorbance at 280 n m and assayed for D N A polymerase activity.
Assay of DNA Polymerase D N A polymerase was assayed as described by Stevens and Bryant (1978), except that the pI-I was 7.25, and the M g acetate was at a concentration of 5 m m o l 1-1.
Assay of Deoxyribonuclease Deoxyribonuclease was assayed in a total volume of 3 ml Tris-HC1 buffer (pH 7.1) containing 1 ml enzyme preparation, CaC12 (2 m m o l 1- 1), Mg acetate (6.7 mmol 1- 1) and 1 m g native calft h y m u s D N A . Assays were carried out at 37~ for 30 rain. The reaction was stopped by addition of 10 ml ethanol containing 0.7 tool l - I perchloric acid. Tubes were stored at - 1 5 ~ for I5 to 18 h. The solutions were then filtered through W h a t m a n No. 1 filter paper. The absorbance at 260 n m of the filtrate was measured, using a filtrate from a reaction stopped at time zero on a blank.
Estimation of Protein
Plants
Protein was estimated by the method of Lowry, Rosebrough, Farr and Randall (1951), using bovine serum albumin as a standard.
Pea seeds (Pisum sativum L., c.v. " F e l t h a m First") were surfacesterilised in sodium hypochlorite (2% available chlorine), washed in running water, and planted in moist vermiculite in seed trays. The trays were kept at 2 5 ~ in darkness. Shoot apices were harvested after five days.
Results
Extraction and Purification of Chromatin-Bound DNA Polymerase Shoot apices were homogenised with a pestle and mortar at 2 ~ C in an equal volume of 50 m m o l 1-1 Tris-HC1 buffer, (pH 7.25) containing 250 m m o l 1 1 sucrose, 10 m m o l l ~ mercapto-ethanol, 1 m m o l 1- 1 m g acetate and 1 m m o l 1- 1 Na2EDTA. The homogehate was filtered through 35 g m nylon mesh. The residue was re-extracted with two volumes of homogenisation buffer, which was also filtered through nylon mesh, and the combined filtrate was centrifuged at 2000 x g for 30 min at 2 4 ~ C. The pellet, a crude chromatin preparation contaminated with cell-wall material, was retained. D N A polymerase was solubilized from the crude chromatin preparations essentially as described by D u n h a m and Cherry (1973). The chromatin preparation was resuspended in one to two ml of 5 0 m m o l 1 - 1 Tris-HC1 buffer (pH 7.25), containing
Reproducibility of data Unless otherwise stated, each point in each figure is the mean of triplicate determinations taken from one of several similar experiments. Standard errors of means were between + 1% and + 15%, although the majority fell in the range _+2% to _+8%.
D EAE-Cellulose Chromatography Chromatin-bound DNA polymerase is eluted from DEAE-cellulose columns as a single peak at 0.2 to 0.4 mol 1-1 KC1 (Fig. 1). The exact position of the peak is variable, and the peak itself is also somewhat heterogeneous. These features are not caused by con-
C. Stevens et al. : C h r o m a t m - b o u n d D N A Polymerase from Higher Plants
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Fig. 2. Fractionation of solubilized chromatin-bound D N A polymerase by DEAE-Sephadex chromatography, a Freshly prepared enzyme extract, b Enzyme extract stored at - 1 5 ~ for I0 days prior to chromatography. The arrows in a represent the eleution positions of deoxyribonuclease
DEAE-Sephadex Chromatography pH and Ion Requirements The enzyme is eluted from columns of DEAE-Scphadex as two peaks (Fig. 2a). The smaller peak (CI), representing 15 to 25% of the total activity, does not bind to the column, and is eluted in the washing buffer. The major peak (CII) is eluted at 0.3 to 0.4 mol 1- ~ KCI. The extent of purification achieved, compared with crude chromatin preparations, is usually 5 to 10-fold for CI and 35 to 40-fold for CII. Freezing the enzyme for 15 to 20 h prior to column chromatography has no effect on the elution profile. This contrast markedly with the behaviour of DNA polymerase-c~ (Stevens and Bryant, 1978). However, long-term storage (nine days or more) of enzyme extracts at - 1 5 ~ prior to column chromatography results in the appearance of a third peak of activity (CIII) at 0.55 tool 1-2. KC1 (Fig. 2b). The presence of CIII is correlated with a decrease in the amount of enzyme eluting as CII. This suggests that CIII may be derived from CII. The proportion of enzyme eluting as CI, by contrast, is not changed by long-term storage at - 15 ~ C.
The pH and ion requirements of CI and CII are illustrated in Figures 3, 4 and 5. The data are expressed in terms of relative enzyme activity, in order to compare preparations of different specific activities. CI exhibits no clear magnesium optimum (Fig. 3a), although there is an ill-defined peak at 18 mmol 1-1. CI exhibits a manganese optimum of 1 mmol 1-1 and manganese is very much preferred to magnesium. CII has a magnesium optimum of 4 mmol 1- 1 and a manganese optimum of 1.5 mmol 1 ~; magnesium is preferred to manganese (Fig. 3 b). The KC1 optimum of CI is 150 mmol 1- 1, although activity is also high at 75 mmol 1-1 (Fig. 4). CII is most active at 200 mmol I-1 KC1. Both enzyme populations show sharp pH optima, at 7.3 for CI and 7.1 for CII (Fig. 5). Thus, in general characteristics, the enzyme populations represented by both CI and CII resemble crude enzyme preparations (Robinson and Bryant, 1975; Stevens, 1976), with the exception of the response of CI to manganese and magnesium.
116
C, Stevens et al.: C h r o m a t i n - b o u n d D N A Polymerase from Higher Plants
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Fig. 5. Effects of p H on the activities of CI and CII
Sensitivity to N-Ethyl-Maleimide
than crude preparations and much less strongly than polymerase-c~ (Stevens and Bryant, 1978). Surprisingly, desPite the very similar inhibition curves for CI and CII, the two populations show differing requirements for SH-groups in the assay medium. In contrast to other enzyme preparations, the single peak of activity obtained by DEAE-cellulose chromatography is stimulated by N-ethyl maleimide. There is thus an inverse correlation between the purity of the enzyme preparation and its sensitivity to N-ethyl maleimide.
The sensitivity of the enzyme to the SH-group alkylating reagent, N-ethyl maleimide, has been determined for crude enzyme preparations, for peaks CI and CII obtained by DEAE-Sephadex chromatography, and for the single peak of activity obtained by DEAEcellulose chromatography (Fig. 6). Crude enzyme preparations are strongly inhibited by N-ethyl maleimide (Fig. 6a). CI and CII (Fig. 6b) are inhibited to the same extent as each other, although less strongly
C. Stevens et al. : Chromatin-bound DNA Polymerase from Higher Plants
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Fig. 6a-c. Effects of N-ethyl maleimide (in the absence of mercapto-ethanol) on the activity of chromatin-bound DNA polymerase. a Crude enzyme preparations, b CI and CII obtained by DEAE-Sephadex chromatography. The arrows from t and II indicate the curves obtained with CI and CII respectively, c The single peak of activity obtained by DEAE-cellulose chromatography. In panel (c) the data are means from five separate experiments, each with single or duplicate samples. In panels (a) and (b), replication was as for Figures 1-5. In each panel, the dotted line represents the level of activity obtained in the absence of N-ethyl maleimide, and in the presence of mercapto-ethanol
Sensitivity to Phosphonoacetate P h o s p h o n o a c e t a t e inhibits certain D N A polymerases by binding to the p y r o p h o s p h a t e binding site (Leinbach et al., 1976). In cultured m a m m a l i a n and avian cells, it inhibits the activity o f virus-induced D N A polymerases at very low inhibitor concentrations ( M a o and Robishaw, 1975; Leinbach et al., 1976). In m a m m a l i a n cells, D N A polymerases-~ and -/3 are not inhibited by p h o s p h o n o a c e t a t e . In avian cells, polymerase-/~ is resistant whilst polymerase-c~ is inhibited, albeit by concentrations ten-fold higher than those needed to inhibit the virus-induced polymerases. We have determined the sensitivity o f chromatinb o u n d D N A polymerase to p h o s p h o n o a c e t a t e with crude enzyme preparations and with the single peak of activity obtained by DEAE-cellulose c h r o m a t o graphy. F o r comparison, we have carried out similar experiments with the soluble D N A polymerase-c~ described previously (Stevens and Bryant, 1978). F o r b o t h c h r o m a t i n - b o u n d D N A polymerase and D N A polymerase-c~, crude and partially purified enzyme preparations respond similarly to the inhibitor. Both the c h r o m a t i n - b o u n d enzyme and polymerase-c~ are inhibited by p h o s p h o n o a c e t a t e , although polymerase is m o r e sensitive than the c h r o m a t i n - b o u n d polymerase (Fig. 7). The concentration of p h o s p h o n o a c e tate required to give 50% inhibition is ca. 65 ~tmol 1 1 for polymerase ~ and ca. 155 pmol 1-1 for the
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Fig. 7. Effects of phosphonoacetate on the activities of chromatinbound DNA polymerase ( e - e - e ) and soluble (~) DNA polymerase (o-o 9 Data prepared as in Figure 6c
118
C. Stevens et al. : Chromatin-bound DNA Polymerase from Higher Plants
chromatin-bound polymerase. These concentrations of inhibitor are significantly higher than those required to give 50% inhibition of the avian polymerase-c~, and 10 to 50-fold higher than the concentrations required to give 50% inhibition of virus-induced polymerase, (Mao and Robishaw, 1975; Leinbach et al., 1976).
Nuclease Activity Chromatin preparations contain deoxyribonuclease activity (Jenns and Bryant, 1978). The nuclease is solubilised from chromatin with the polymerase, and the majority of the nuclease activity co-chromatographs with the polymerase on columns of DEAEcellulose. On columns of DEAE-Sephadex, two peaks of nuclease activity are obtained (Fig. 2a). Neither is coincident with a peak of DNA polymerase activity, strongly suggesting that the DNA polymerase does not possess deoxyribonuclease activity.
Template Preferences CI and CII have been assayed with only a limited number of templates (Table 1). Both forms of the enzyme show a preference for activated DNA. However, CI achieves relatively high rates of reaction with non-activated templates, and in fact does no distinguish between double-stranded and single-stranded DNA. These relatively high reaction rates with nonactivated templates may be caused by the presence of small amounts of nuclease, since although peak I of nuclease activity elutes from DEAE-Sephadex before CI (Fig. 2), the two activities in fact overlap. CII, by contrast, achieves relatively high rates with single-stranded DNA, but only very low rates with native DNA. The template preferences of the single peak of activity obtained by DEAE-cellulose chromatography are intermediate between those of CI and CII.
Table 1. Template preferences (with calf-thymus DNA) of CI and
CII. For each enzyme preparation, the activity obtained with activated DNA has been taken as 100 Template
Relative enzyme activity CI
Activated DNA 100 Denatured DNA 77.6 Native DNA 78.2
CII 100 46.2 15.6
Discussion
In this paper, we have demonstrated that the chromatin-bound DNA polymerase from pea seedlings exhibits multiple forms when chromatographed on columns of DEAE-Sephadex. Two peaks, CI and CII are usually obtained, and a third, CIII, is generated from CII by long-term storage at - 1 5 ~ C. We have not been able to demonstrate any interconvertibility between CI and CII but the general similarity of their properties strongly suggests that they represent different populations of the same enzyme. The exact relationship between CI, CII and CIII remains to be established, but it is likely that they represent different aggregation states of the native enzyme, such as has been observed for the chromatin-bound enzyme of a number of different cultured human cell lines (Srivastava, 1974; Wang et al., 1975). The properties of the chromatin-bound polymerase from pea seedlings are very different from those of the soluble DNA polymerase-c~ (Stevens and Bryant, 1978) and in many respects resemble the properties of DNA polymerase-/~ of vertebrates. However, a number of investigators have stated that higher plants do not possess a chromatin-bound DNA polymerase analogous to polymerase-/? (Chang, 1976; Gardner and Kado, 1976; McLennan and Kerr, 1977). Two criteria, in addition to sub-cellular location, are generally taken as indicative of DNA polymerase-/~. These are low molecular weight (ca. 5 x 104 daltons) and insensitivity to SH-group reagents, such as N-ethyl maleimide. We strongly suggest, on the basis of these criteria, that higher plants do possess DNA polymerase-/~. Firstly, the enzyme activity discussed in this paper represents a nuclear enzyme, being detected both in chromatin preparations and in isolated whole nuclei (Stevens, 1976). Activity is never observed in the 34,000 x g supernatant, and therefore the activity is not soluble. Similar chromatin-bound DNA polymerases exist in Tradescantia (Wever and Takats, 1970), soybean (Leffler et al., 1971), tobacco (Srivastava and Grace, 1974) and beet (Tymonko and Dunham (t977). Secondly, there is clear evidence from other investigators that these chromatin-bound enzymes are of low molecular weight. The chromatinbound enzyme from tobacco has a sedimentation coefficient of 3.4 S (Srivastava and Grace, 1974). The chromatin-bound DNA polymerase of beet also has a sedimentation coefficient of 3.4 S, with a molecular weight of 5.02 x 104 daltons (Tymonko and Dunham, 1977). These estimates of size are very similar to those obtained for DNA polymerase-]~ in vertebrates. Unfortunately, we have not been able to obtain reproducible molecular weight estimates for the chromatinbound polymerase from pea seedlings, because the
c. Stevenset aI. : Chromatin-bound DNA Polymerasefrom Higher Plants enzyme is subject to aggregation-disaggregation phenomena. However, experiments to establish the optimal conditions for determination of molecular weight are now in progress. Thirdly, the chromatin-bound enzymes from higher plants are not sensitive to the SH-group reagent, N-ethyl maleimide. In beet, the 3.4 S activity is inhibited only 1% by 0.75 mmol 1- 1 N-ethyl maleimide (Tymonko and Dunham, 1977). In our experiments, the sensitivity to N-ethyl maleimide is related to enzyme purity. The least pure preparations are sensitive, whilst the preparations with the highest specific activity are actually stimulated by Nethyl maleimide. Similarly confusing data have been obtained with DNA polymerase-fl from mammals. In several different types of cultured human cells the sensitivity of polymerase-fi to N-ethyl maleimide varies with the purity of the enzyme (Srivastava, 1974). The nuclear DNA polymerase from rat liver is slightly stimulated by N-ethyl maleimide at concentrations below 0.5 mmol 1-1 but is strongly inhibited by another SH-group reagent, p-hydroxymercuribenzoate (Zunino et al., 1975). It is apparent that as a criterion for identification of polymerase-fi, resistance to SHgroup alkylating agents must be with care. However, the data obtained for higher plant chromatin bound DNA polymerases are at least consistent with the view that they may be classified as DNA polymeraseIn addition to these major criteria, several other features of the chromatin-bound DNA polymerase from pea seedlings reflect its similarity to DNA polymerase-fi of vertebrates. Firstly, the enzyme has a high KC1 optimum, in marked contrast to the soluble DNA polymerase-~ from the same plant (Stevens and Bryant, 1978). Secondly, the major form of the enzyme clearly prefers magnesium to manganese. This contrasts markedly with DNA polymerases from a number of lower eukaryotes, including the unicellular alga, Euglena (McLennan and Keir, 1975). Thirdly, we can detect no nuclease activity co-chromatographing with either CI or CII, although CI may contain low levels of nuclease activity because of the overlap of the nuclease and polymerase peaks. Lack of nuclease activity is a feature of all DNA polymerases from vertebrates, and also of the DNA polymerase-c~ of pea seedlings (Stevens, Jenns and Bryant, 1976). By contrast, in several lower eukaryotes, at least one of the DNA polymerases possesses nuclease activity (McLennan and Keir, 1977; Ross and Harris, 1978). Fourthly~ as with the chromatin-bound DNA polymerase-fi of vertebrates, changes in the activity of the chromatin-bound DNA polymerase of pea show little or no correlation with DNA synthesis (Jenns and Bryant, 1977). Although the properties of the chromatin-bound
119
DNA polymerases of higher plants clearly characterise them as DNA polymerase-fl, the enzymes from plants do show two minor differences from the enzymes from vertebrates. Firstly, in pea (this paper), Tradescantia (Wever and Takats, 1970) and soybean (Leffler et al., 1970), the pH optimum of polymerase-fl is neutral or near neutral. In most vertebrates, the pH optimum of polymerase-fi is markedly alkaline (Brun and Chapeville, 1977). Secondly, in our experiments, DNA polymerase-fl is inhibited by phosphonoacetate. However, the polymerase-fi is markedly less sensitive than polymerase-~ from the same plant, and further, the concentrations of phosphonoacetate required for significant inhibition are very high (cf. Mao and Robishaw, 1975; Leinbach et al., 1976). Thus, this apparent difference between DNA polymerase-fi of pea seedlings and DNA polymerase-fi of vertebrates may be quantitative rather than qualitative. Consideration must also be given to the failure of other investigators to detect a chromatin-bound DNA polymerase-fl in higher plants. Chang (1976) used the embryo of a quiescent seed, namely wheat germ, in her experiments. Ungerminated wheat germ possesses only very low levels of total DNA polymerase activity (Mory et al., 1975) and by analogy with other seeds (Robinson and Bryant, 1975) it is likely that polymerase-fi makes up only a small proportion of the total activity. The pH used in Chang's experiments was 8.0; this is higher than the pH optimum for most higher plant chromatin-bound polymerases (see above). Indeed, for the DNA polymerase-fi from pea described here, activity at pH 8.0 is only 30-50% of that at pH 7.1 to 7.3. Further, Chang used relatively crude extracts for enzyme assays which may well give misleading results when the effects of Nethyl maleimide are determined (see above). In the work of Gardner and Kado (1976), no direct assays were made of chromatin preparations. Instead, chromatin was solubilized by the method of Chang (1973), and the solubilized extract was assayed for DNA polymerase. Although this solubilization method is clearly effective for calf thymus tissue, we have found that it yields, at best, only 5-10% of the chromatinbound DNA polymerase activity from pea seedlings (Stevens and Bryant, unpublished data), It therefore seems likely that Gardner and Kado failed to recover chromatin-bound polymerase in their experiments. Finally, it must be emphasised that in dividing plant cells, DNA polymerase-fl activity makes up only between 1 and 25% of the total DNA polymerase (Srivastava and Grace, 1974; Robinson and Bryant, 1975; Jenns and Bryant, 1977). Unless attempts are made to separate the two enzymes prior to assay, it is quite possible for the DNA polymerase-fi to be masked by polymerase-e.
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C. Stevens et al. : Chromatin-bound DNA Polymerase from Higher Plants
We thank the Science Reasearch Council for the award of a research studentship to C.S., and Shell Research Ltd. for financial support. We are grateful to Dr. V.L. Dunham for helpful discussion and to Dr. A.G. McLennan for a gift of phosphonoacetic acid.
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