J. Mol. Bid. (1976) 93,219-236

The Distributive Nature of Enzymatic DNA Synthesis LUCY M. S. CUCZJ

Department of Biochemisty Univemity of Connecticut Health Center Farmington, Conn. 06032, U.S.A. (Received25 June 1974, and in revised jorrn 13 November1974) The consequences of enzyme and template interaction were examined by several independent methods in the replication reactions catalyzed by calf thymus low molecular weight DNA polymerase, calf thymns high molecular weight DNA polymerase, and Emherichia coli polymersse I. All methods used support a distributive, rather than processive, mechanism for enzyme in the replication of homopolymer templates dn vitro. First, addition of an excess of initiated poly(dC) template to an ongoing poly(dA) replication reaction results in immediate cessation of dTTP polymerization. Second, the kinetics of monomer and initiator incorporation in reactions where a large number of initiated template molecules are available to each enzyme molecule show early incorporation of all initiators followed by simultaneous replication of the total population of template molecules. Third, alkaline sucrose gradient analysis of the products formed at various stages during replication show simultaneous growth in product chain lengths. Fourth, analysis of products formed when an average of one to two nucleotides are added at the end of the growing chain, in reactions having a molecular ratio of template to enzyme of about 900, show that the enzyme can dissociate from the replicating template after a single addition. Incressing the ionic strength of the reaction mixture, to decrease the secondary interactions between the enzyme and the template, results in nearly random interaction of the enzyme and the template. The results from this study suggest that translocation of template chain during replication is not an obligatory function of purified DNA polymersses. The possible involvement of other proteins required for DNA replication in V&O in the interaction of DNA polymerase and DNA is discussed.

1. Introduction Enzymatic synthesis of DNA describes a rather complex and important process in over-simplified terms. It is quite obvious that DNA polymerase is only one of the many components required in the replication of DNA in vivo (Epstein et d., 1963; Bollum, 1963; Alberts, 1973; Goulian, 1971; Klein & Bonhoeffer, 1972). It is not appropriate to attribute the characteristics of in vivo replication to simple enzyme systems without a logical experimental analysis. Once this analysis is performed, however, the behavior of cellular or reconstructed systems becomes more comprehensible. Even though the function of DNA polymerase in DNA replication seems to be restricted to elongation of the product chain under the direction of the template chain, detailed mechanistic studies are difficult to carry out because of the complexity and multiplicity of substrates required for heteropolymer replication. 16

@l@

220

L.

M. 5. CHANC4

The reactions of Eschwichia coli polymerase I have been studied in some detail by Kornberg and co-workers (Kornberg, 1969). The mechanism of the polymerization catalyzed by this enzyme is complicated by the presence of two associated nuclease activities. The mammalian DNA polymerases, however, have no associated nuclease activity (Chang & Bollum, 1973). The requirements for the polymerization function of the enzyme can be easily established for the mammalian enzymes (Bollum, 1967; Chang, 1973a). These requirements include a template polydeoxynucleotide, an oligodeoxynucleotide complementary to the template, a complementary deoxynucleoside triphosphate, a divalent cation and a suitable ionic environment. The availability of model template systems (Bollum, 1964) composed of homopolymers and homo-oligomers has further reduced the complexity of the polymerization reaction. In these systems the DNA polymerase reaction is a simple bisubstrate reaction; the substrates are initiated template and divalent cation: dNTPt, and the products are pyrophosphate and the initiated template with one nucleotide added at the 3’terminus of the initiator chain. Since the polymer product is recycled as a substrate in the reaction, it has frequently been assumed that this product remains associated with the enzyme throughout replication. The analysis presented here demonstrates that this assumption is incorrect for E. coli polymerase I, high molecular weight and low molecular weight DNA polymerases from calf thymus. The polymerization reaction catalyzed by E. coli DNA polymerase I has been described as a two-step process in which the initiator chain is first elongated by nucleophilic attack on the a-phosphoryl group of the incoming dNTP by the 3’hydroxyl group of the initiator chain, followed by translocation of the template on the enzyme surface to move the 3’-hydroxyl group of the newly added nucleotide to the initiator terminus binding site (Kornberg, 1969). Enzyme-bound Zn has been proposed to be involved in the translocation of the polynucleotide on the enzyme surface (Slater et al., 1972). An enzyme obeying this model should replicate a population of template molecules in a processive manner. In a processive replication reaction, three polynucleotide populations should be present in the system during replication ; these populations are unreplicated templates (templates), templates in the process of replication (intermediates), and completely replicated templates (products). In reactions with a high molecular ratio of template to enzyme, the amount of material in intermediates should be negligible at any stage of reaction and the template molecules should be gradually converted to full product molecules as replication proceeds. Examination of this model with three different DNA polymerases demonstrates that translocation is not part of the simple DNA polymerasecatalyzed reaction. Under, certain reaction conditions, the enzyme interacts with the template in a near statistical manner; the enzyme molecule dissociates from the template after each polymerization step, and further reaction requires the reassociation of the template at the proper binding sites on the enzyme surface.

2. Materials and Methods (a) Substrates Polydeoxynucleotides, oligodeoxynucleotides and dNTPs were prepared &8 previously described (Chang & Bollum, 1973; Chang, 1973a). Polydeoxynucleotides with known chain t The abbreviationa used are those approved by CBN of IUPAC-IUB ia used w en abbreviation for potassium phosphate.

and this Journal.

KP,

ENZYMATIC

DNA

SYNTHESIS

221

lengths, used as standards for chain length determinations, were synthesized using terminal deoxynucleotidyltransferase and radioactive oligodeoxynucleotides as initiators (Bollum, 1966). The chain length of each of the final products was obtained by determination of total nucleotide concentration by optical density measurement and determination of the concentration of the 5’-ends of the polymer solution by radioactivity measurement. Radioactive dNTPs were purchased from Schwarz-Mann Research and New England Nuclear Corp. Bovine serum albumin andpoly(rA) were purchased fromMiles Laboratories. All other reagents were commercial grade. (b) Enzymes Calf thymus low molecular weight DNA polymerase was prepared as previously described (Chang, 1973b). The enzyme preparation is essentially homogeneous. The calf thymus high molecular weight DNA polymerase was prepared from the soluble extract of calf thymus glands essentially as described by Yoneda & Bollum (1966) followed by chromatography on DEAE-cellulose. Although the high molecular weight DNA polymerase enzyme is not homogeneous, no nuclesse activity can be detected in this preparation (Chang & Bollum, 1973). E. coli polymerase I containing about 90% enzyme protein was generously supplied by Dr L. A. Loeb, Institute for Cancer Research, Fox Chase, Philadelphia, Pa, U.S.A. The enzyme activities were assayed using d(pA)G d(pT),, as template and dTTP aa monomer. In the usual reaction mixture, the nucleotide concentration of poly(dA) was O-1 IIIM, the nucleotide concentration of d(pT),, was 0.01 mM, and the concentration of [meth$-3H]dTTP was 0.2 mM (spec. act. of the isotope used ranged from 10 to 100 cts/min per pmol). Calf thymus low molecular weight DNA polymerase reactions were carried out in 50 m&r-Ammediol-Cl buffer (pH 8.6), 0.1 M-NaCl, 1 m&r-2-mercaptoethanol, 50 pg bovine serum albumin/ml and 0.5 mM-MnCl,. Calf thymus high molecular weight DNA polymersee reactions contained 20 mm-KP, (pH 7.0), 1 m;n-2-mercaptoethanol, 50 .ug bovine serum albumin/ml and O-5 m&r-M&l,. E. coli polymerase I reactions were carried out in 40 mMTris*HCl buffer (pH 8-O), 1 m&r-2-mercaptoethanol, 60 pg bovine serum albumin/ml and 8 mM-MgCl,. Using these conditions, the specific activity of the enzymes used were determined to be 880,000, 9900 and 620,000 units/mg of protein for calf thymus low molecular weight DNA polymerase, calf thymus high molecular weight DNA polymersse and E. coli polymer&se I, respectively. One unit is defined aa the amount of enzyme required for polymerizing one nmole of dTTP/h at 35°C. The polymer, oligomer, and enzyme concentrations used in experiments to be described varied according to the specific purposes of the particular experiment. The concentrations of polymer and oligomer in the remainder of this study are expressed in molecular concentrations. (c) Alkaline sucrose gradient analyka of the products of DNA polymeraee reactions The products of poly(dA) replication at different stages of enzymatic replication were analyzed by alkaline sucrose gradient centrifugation. The average chain length of the products was determined by comparison with the sedimentation behavior of polymers of known chain lengths using identical conditions for analysis. The polymers used as standards in this study are [14C]d(pT)sd(pT)G, P4WW%d(pT),8a and V4Q4pT)6d(pT)so4. DNA polymerase reactions were terminated by the addition of EDTA, NaCl and NaOH to final concentrations of 20 mu, 0.2 M and 0.2 M, respectively. The samples were centrifuged in linear gradients of 5% to 20% sucrose (w/w) in 10 mM-EDTA, 0.2 M-NaCl and O-1 M-NaOH for 20 h at 40,000 revs/mm in the SW50.1 rotor at 4°C. The gradients were then fractionated into equal volume fractions from the top of the gradient tubes with a Buehler Densi-flow apparatus. The pH of the gradient fractions was adjusted to about 8.8 with 1 M-Tris.HCl (pH 7.0), and poly(rA) was added to each fraction to ensure complete acid-precipitation of poly(dT) in the fraction. A portion from each fraction was processed on a Whatman GF/C disk and counted in the liquid scintillation counter as previously described (Chang, 1973a). The position of the template nucleotide (poly(dA)) in the gradient was determined by spectrophotometric analysis of the gradient fractions from a gradient tube containing only the template polymer. All gradients were normalized to a fraction of the total gradient with 0 representing the top of the gradient and 1 representing the bottom of the gradient.

L. Y. 8. CHANG

a22

(d) An&& of the poduct% of DNA po&me~~e reactiow by gd ji&& on Sephadux a?‘5 and column ohrmtztography on DEAE-celluhe The sizes of the products at early stages of poly(dA) replioation were determined by f&ctionetion on a Sephadex G76 column. DNA polymeram reactions (0.26 ml) were terminated by the addition of EDTA, N&l and NaOH and separated on a Sephadex G76 column (O-6 am x 110 cm) equilibrated with 0.1 N-N&OH, O-2 M-N&~ and 1 mM-EDTA. Radioaotivities in the column fractions (0.226 ml) were determined by counting B portion of each fraction in Aquasol (New England Nuclear Corp.) after acidifkation of the sample with 2 N-HCl. The template eluted from the column in the void volume and was determined by spectrometric amdysis of the column fractions. In order to analyze the produots of poly(dA) replication by column chromatography on DEAR-cellulose (DE62, Whatman), preliminary sepamtion of the bulk of the products from the template was carried out on a Sephadex G76 column at alkaline pH as desoribed above. A one-ml reaction mixture was fractionated on a Sephadex G76 column (0.8 cm x 120 cm), and 0.46~ml fractions were collected. The product pool from the Sephadex column was adjusted to pH 4.7 with 1 M-acetic acid, and then loaded onto a DE62 cellulose aohmm (0.3 cm x 24 cm) equilibmted with 0.2 M-N&~ in 0.02 ~-sodium acetate buffer (pH 4.7). The column was washed and the oligodeoxynucleotide were eluted from the in each column with a 400-ml linear N&l gradient from 0.2 M to 0.4 M. Radioactivities fraction (1.4 ml) were determined by counting under double isotope counting oonditions in Aquasol.

3. Results (a) Template competition experiments To examine whether template

molecule

a DNA polymerase molecule will completely replicate one before starting on another template molecule, the effect of the

addition of initiated poly(dC) to a reaction in the process of poly(dA) replication was studied. In these experiments, poly(dA) replication is allowed to proceed to about 10% of the available template nucleotide so that the enzyme has a head start on the poly(dA) template system prior to the addition of competing template system (initiated poly(dC)). If a processive polymerization is in progress one would expect to observe some continuing replication of the poly(dA) template immediately after the addition of the competing template. If an enzyme is distributive in nature, that is if the enzyme dissociates from the template during replication, one would expect to observe immediate inhibition of the poly(dA) replication. The effect of the addition of initiated poly(dC) in the poly(dA) replication reaction catalyzed by E. coli polymerase I is shown in Figure 1. The template system used for E. wli polymerase I was d(pA)G*d(pT)Eo at an initiator to template ratio of 0436. The molecular ratio of enzyme to initiator is about one. The data in Figure 1 clearly show that dTTP polymerization ceases immediately after addition of the competing template, suggesting that the enzyme molecules in the process of replicating poly(dA) at the time the competing template was added did fall off from the poly(dA) template. When a significant portion of the competing templates was replicated, dTTP polymerization started again but at a slower rate compared to the control poly(dA) replication. It wuld certainly be argued that the atllnity of the E. co.6polymerase I for poly(dC) template may be many-fold higher than the affinity for poly(dA) template and that the difference in the afhnities of the enzyme for the different template systems might account for the results obtained. This criticism can be eliminated if similar results were obtained with the oalf thymns low moleoular weight DNA polym~raae. The

ENZYMATIC

30

DNA

SYNTHESIS

60 90 Reaction time (minl

223

120

FIG. 1. Template competition experiment. E. wli polymerase I. This Figure shows the effect of the addition of d(pC)3gi*d(pG)s to the d(pA)s*d(pT)rs replicetion reaction. The reactions were carried out at 35OC in a total vol. of 0.26 ml. The amount of enzyme used/reaction was 4.5 pmol. Reaction 1, containing 5-7 pmol of d(pA& and 4.9 pmol of d(pT)G, was started by the addition of the enzyme. Reaction 2, containing also 5-7 pmol of d(pA)a and 4.9 pmol of d(pT)z, was also started by the addition of the enzyme except that a mixture containing 250 pmol d(pG)s and 37 pmol d(pC)cs was added after 30 min of inoubation. Reaotion 3, containing 250 pm01 of d(pGh and 37 pmol dWki, was started by the addition of enzyme. The concentrations of other components in the reaction were 40 mr+Tris*HCl buffer (pH 8.0), 1 maa-2-mercaptoethanol, 0.1 ~-N&cl, 8 mna-MgCls, 0.2 maa-[2-iV]dTTP, 0.1 mM-[%sH]dGTP and 50 pg bovine serum albumin/ml. For eaoh reaction, 20-d portions were removed at various times and analyzed. The open symbols represent the results for oontrol poly(dA) and poly(dC) replication reactions (reactions 1 and 3). The closed symbols represent the results obtained by addition of d(pC)FO*d(pG)g to the poly(dA) replication reaction (reaction 2).

K, values of the low molecular weight DNA polymerase for various template systems are about the same (Chang, 1973o). The results obtained for the low molecular weight DNA polymerase using identical template systems are similar to those obtained for E. wli polymerase I, even though the progress curves for the low molecular weight DNA polymerase reactions using these lone templates were non-linear. To show that mammalian enzymes are also non-processive with regard to template, a shorter poly(dA) template system was used. To facilitate the manipulations required for this hind of experiment, the reactions were carried out a lower temperature and for a much shorter time. The results on the mammalian enzyme are shown in Figure 2. The addition of poly(dC) template to ongoing poly(dA) replications catalyzed by the mammalian enzymes resulted in an immediate inhibition of poly(dA) replication. The inhibition of dTTP polymerization in the mammalian DNA polymerase reactions by the addition of initiated poly(dC) was not complete. This apparent partial processive nature of the enzymes in these reactions will be discussed in a later section. The results obtained from template competition experiments for all three DNA polymerases show that added template is competitive and thus the enzyme does dissociate and reassociate with template molecules during replication. The results presented in this se&ion are oonsistent and supportive with a distributive nature of

224

L. M. 9. CHANC (0 ) Calf thymus polymerase

3 4 S DNA

( b ) Calf thymus polymarose

6 to 6 S DNA

I

0

60

120

180 Reaction

0 time (s)

60

120

180

FIG. 2. Template competition experiment. Calf thymus DNA polymeraaes. This Figure shows the effeot of the addition of d(pC)G*d(pC), to d(pA)G*d(pThn replioation reaotions. The protoools for the reactions carried out by each of the calf thymua enzymes were the Bame aa desaribed for E. wli polymeraae I reactions except that the amounts of polymers and oligomera used were 17 pmol of d(pA)G, 17 pmol of d(pT),*, 30 pmol of d(pC)E and 332 pmol d(pC)B. The amount of enzyme present in the calf thymua low molecular weight DNA polymeraae reactions was 11 pmol. The amount of enzyme present in the calf thymus high moleoular weight DNA polymerase reaotions w&s 360 enzyme units. Other aomponents and their oonoentrations present in the reaotions were as desoribed in Materials and Methods for the 2 enzymes exaept that O-1 no[8-SH]dCTP W&B also present. The symbols used are the same as in Fig. 1. See Material and Methods and the legend to Fig. 1 for further details.

DNA synthesis catalyzed by all three DNA polymerases examined. More conclusive evidence for the mechanism of the enzymes can be obtained by direct analysis of the products of the replication reactions. (b) Direct analysis of products during poly(dA) replication DNA polymerases cannot initiate new chains and replication of homopolymers occurs only in the presence of complementary oligonucleotides. The extent of bulk polymer replication can be estimated from the amount of complementary dNTP converted into product. If a radioactive oligonucleotide is used as an initiator, the use of the 3’-hydroxyl end of the initiator as the growing point of the replication process can be easily demonstrated (Chang et al., 1972). The kinetics of monomer polymerization reflect the conversion of single-stranded template into doublestranded product. The amount of acid-soluble initiator converted into acid-insoluble product reflects the number of growing chains involved in this process. In a DNA polymerase reaction where the synthetic process is not complicated by degradative processes, the amount of initiator incorporated corresponds to the number of chains growing in the reaction and the average chain length of the product chains can be calculated by the monomer to initiator ratio at any stage of the replication. For an enzyme which is processive in nature, one would expect to see a gradual incorporation of initiator, while the chain length of the product calculated from monomer to initiator incorporation should remain relatively constant throughout the reaction.

ENZYMATIC

DNA

225

SYNTHESIS

In other words, the chain length calculated by this analysis would be constant and about the same as the chain length of the template. For distributive chain growth, one would expect rapid incorporation of initiator molecules early in the replication followed by a gradual increase in the average chain length of the products. The poly(dA) replication reactions for the three DNA polymerases carried out using[1414pT)Iaand [naethyL3H]dTTP are shown in Figure 3. The ratio of enzyme to initiator used was set at about 100. Figure 3 illustrates the kinetics of initiator and

500

:a)

lb1

0 20 40 60 80

;;-20

Calf thymus 6to8S

(cl

:alf thymus 3.4 S

E co/r

polymerose

I

1

2. 400 ', 300 5 2oc 5 u

100 0

40 60 80

Reaction

time

0 20 40 60 80

(mm)

Fra. 3. Direct analysis of ah&in growth in DNA polymeraaa reactions. The time course of monomer and initietor incorporation ~88 measured in DNA polymerase reactions containing 118 pmol of d(pA)z, 130 pmol of [‘*C]d(pT)la and 0.2 mM-[methyL3H]dTTP. The amounts of enzyme present in the me&ions were 70 enzyme units, 2.2 pmol, and 1.1 pmol for calf thymus high molecular weight DNA polymerase, calf thymus low molecular weight DNA polymerese and E. coli polymerase I, respectively. The chain lengths were calculeted by the ratios of monomer to initiator in the products. See Materials and Methods for further details.

monomer incorporation and the calculated average chain lengths of the product chains. For both mammalian enzymes, more than 80% of the initiator molecules present in the reaction are found to be incorporated into products at early stages of replication (Fig. 3(a) and (b)). For the high molecular weight calf thymus enzyme, all initiator molecules were in the products when the template nucleotide was about 30% replicated (Fig. 3(a)). For the low molecular weight DNA polymerase reaction, an initial burst of initiator incorporation was followed by slow and gradual incorporation of those remaining. The data shown in Figure 3 were obtained for reactions in which the initiator to polymer ratio was about one. Increasing the initiator to polymer ratio in the reactions to two by doubling the amount of initiator resulted in essentially the same type of kinetics for initiator incorporation except that extent of initiator incorporation was about doubled for both mammalian enzymes. Since neither mammalian enzyme has any associated nuclease activity the average chain lengths of the products can be calculated directly from the monomer to initiator ratio in product at any stage of the reaction. It is clear that at a high molecular ratio of

L. M. S. CHANa

226

template to enzyme most template molecules are replicated simultaneausly. When a similar experiment was carried out with E. CC&polymeraae I (Fig. 3(c)), the initiator molecules were inaorporated early in the reaction suggesting that each molecule of polymerase I also replicates a large number of template molecules simultaneously. With E. wli polymerase I the radioactivity associated with the initiator molecules, however, decreased with increased incubation time. The loss in intiator radioactivity is probably due to the actions of the two associated nucleases of E. coli polymerase I (Kornberg, 1969). Due to the degradative activities associated with the enzyme the average chain length of the products of the E. wli polymerase I reaction cannot be calculated directly and must be estimated by comparison with standards. (c) Sedimentation analysis of the products To substantiate further the fact that DNA polymerases replicate template molecules distributively, the average chain lengths of the products at various stages of replication were displayed on alkaline sucrose gradients. Poly(dA) replication products were synthesized in reactions having a high molecular ratio of template to enzyme as described in the previous section. A distributive growth of the product chains should display a gradual increase in average chain length with extent of replication. The product analyses of the E. wli polymerase I reactions are complicated by the fact that the growing chains do not remain intact. The average chain length of the

0.4 0.2 5

0

2

I.5

.-6 c2 I.0 \ k 0.5 6 Twl Y

0 3 2 I

0

0.2

0.4

0.6

Fraction

0.8

I-O

no.

FIG. 4. Product analysis on alkaline sucrose gradients. E. coli polymerase I. The reaction conditions used for the synthesis were as described in Fig. 3. The extents of replication were 17%, 63% and 90% for results shown in (a), (b) and (o), respeotively. The arrow indicates the position of the template poly(dA) in the gradient. Gradient results were normalized with 0 representing the top of the gradient and 1 representing the bottom of the gradient. See Materials and Methods, the text, atid the legend to Fig. 3 for furthm details.

ENZYMATIC

DNA

SYNTHESIS

227

products cannot be obtained directly by monomer to initiator ratio in the products, but a rough estimate of product chain length can be obtained from the extent of replication. The estimation is made by assuming distributive growth of all product chains, each template chain contains one product chain, and no degradation of the growing chains. The alkaline sucrose gradient profiles of the products of poly(d$) replication catalyzed by E. coli polymerase I when the extents of replication were 17%, 530/b and 90% are shown in Figure 4. The estimated average chain lengths for the products at these stages of replication are 128, 375 and 624. The average chain lengths of the bulk of the products for these samples were measured to be 115, 320 and 480. The shorter chain length of the bulk of the products of E. coli polymerase I reactions is readily explained in terms of the degradative activities associated with the enzyme, and the presence of relatively high molecular weight product chains formed in the reactions. The material at the bottom of the gmdients (Fig. 4) has an average chain length longer than the poly(dA) template. This material was analyzed further in replication reactions carried out using [14C]poly(dA), non-radioactive d(pTh, and [methyL3H]dTTP, and the reaction products were displayed on alkaline sucrose gradients at shorter centrifugation time. The results showed that the high molecular weight products were formed throughout replication, that this material is poly(dT) (not covalently linked to poly(dA)), and that the molecular weight of this poly(dT) is heterogeneous. The long poly(dT) chains are probably formed by the continuing action of the enzymes at the end of the template and product chains with simultaneous “creeping” of the template and product chains. Under the conditions examined it is a minor product. When the poly(dA) replication products of calf thymus high molecular weight DNA polymerase are analyzed on alkaline sucrose gradients, the average chain length of each sample can be calculated by the monomer to initiator ratio. Figure 5 shows the profile of the early and late replication products of the high molecular weight enzyme on alkaline sucrose gradients. The extents of replication for these samples calculated by percentage of template nucleotide replication are 1494, 510/, and 892:. The average chain lengths of the products in these samples calculated by incorporated monomer to initiator ratio are 125, 330 and 490. The gradient results clearly show that the bulk of the initiators were incorporated into products early in the replication and the average chain lengths of the products increased with increased replication. The average chain lengths of the bulk of the products were measured to be 145, 480 and 650 for the three samples analyzed. Although the bulk of the initiators were incorporated early in the reaction, the radioactivity associated with the initiators appears in a major population of poly(dT) of average chain length of 650 (indicated by the letter A in Fig. 5(c)) and a minor population of poly(dT) of average cha,in length of 190 (indicated by the letter B in Fig. 5(c)) at later stages of replication. The two populations of product molecules and the somewhat longer than expected chain lengths found for the bulk of the products suggest tha,t some biases do exist in the interaction of the enzyme and the template. The alkaline sucrose gradient profiles of the products of poly(dA) replication catalyzed the calf thymus low molecular weight DNA polymerase are shown in Figure 6. The extents of replication of the samples are 5%, 14%, 35:/, and 920io. The average chain lengths of the products for these samples are 98, 120, 195 and 386 calculated by the ratio of monomer to initiator incorporation. The observed average chain lengths by alkaline sucrose gradient analyses are estimated to be 150. 180. 260

228

L. M. 8. CHANC)

0

0.2

0.6 0.4 Fraction no.

0.8

I.0

Fro. 6. Product analysis on alkaline suorose gradient. Calf thymus high molecular weight DNA polymemse. The conditions used for synthesis were the same aa desoribed in the legend to Fig. 3. The extents of replioation were 14%, 61% and 89% for results shown in (a), (b) and (c), respeatively. The arrow marks the position of template poly(dA) in the gradient. The letters A and B in (c) indicate the 2 populations of produat ohains. See Materials and Methods, the text and the legend to Fig. 3 for further details.

and 420 for these samples. The distributions of the molecular weights of the products of the low molecular weight DNA polymerase reactions are much broader than the distributions of the products observed for the other two enzymes, and the measured chain lengths of the bulk of the products are somewhat longer than expected. Although the results obtained in the product analyses are less than perfect for a purely random interaction of the enzyme with the growing point of the initiated template, it is clear that each DNA polymerase molecule mn replicate many template molecules simultaneously. To accommodate the results of this study one must conclude a generally distributive nature for enzymatic DNA synthesis. The enzyme molecule must dissociate from the template molecule during synthesis to produce random replication distributed over the entire template population. (d) Analysis of early products of poly(dA) replication Although a generally distributive mechanism of DNA replication for all three DNA polymerases can be deduced from the results presented in the previous sections, the exact molecular process for individual steps becomes blurred. This section deals with the question whether the enzyme dissociates from the template after each polymerization process. If the interaction between the enzyme and the template is purely distributive, then the chain length distribution of products should be Poisson.

ENZYMATIC

DNA

229

SYNTHESIS I

I

I

t 3.0 I.5

0 I ’

I

I-

I

0

1 .

3

z E

I.5

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0 3.0

1! r Q ‘;; I.5 ‘;; o_

0

-

3.0 I 5 0 0

0.2

0.6 0.4 Fraction no

08

I.0

FIG. 8. Produot analysis on alkaline sucrose gradient. Calf thymus low molecular polymerase. The conditions used for synthesis were the same as in Fig. 3 except that [‘%]d(pT)ra were present/reaction. The extents of replication were 6%, 14%, 36% results shown in (a), (b), (c) and (d), respeotively. The arrow marks the position poly(dA) in the gradient. See the text, Materials and Methods and the legend to Fig. details.

weight DNA 175 pmol of and 92% for of template 3 for further

The previous sections seem to indicate some bias in the interaction of the template with the mammalian enzymes. The products of poly(dA) replication catalyzed by the mammalian enzymes were found to be somewhat longer than predicted on the basis of random interaction of enzyme and template. At least part of the bias is expected since an enzyme molecule dissociated from a macromolecular substmte may have a greater probability of reassociating with the same template molecule than associating with other template molecules in solution. In addition, both mammalian enzymes form stable complexes with DNA under the ionic conditions used in the experiments described in the previous sections. The processive tendency of the mammalian enzymes may be enhanced by this kind of secondary interaction of the enzyme with the template. If such secondary interactions are the major contributor to the bias observed, an improved size distribution of the replication products might be obtained by increasing the ionic strength in the DNA polymerase reactions in order to minimize secondary interactions. In order to study the distribution of DNA polymerase products at low degrees of replication, an alternative analytical procedure was used. The early products of homopolymer replication reactions were fractionated by gel filtration on Sephadex G75 columns at alkaline pH. In these experiments the molecular ratio of template to enzyme in the reactions was set at about 900, and 14C-labeled initiator and

230

L.

Lo I: 200

[NaCL]:O

[NoCl] - 0.05~ average addition

M

overage addition I.4

[NaCL] = 0.1 M average odditlon

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I.2

4 - 20s ..z z IO% E o-

‘i I50 .. : I+ 5

M. 8. CHANG

100

s .-z t L \ : p.

50

O-

50

60

70 (0)

80

90

60

70

80

90

60

70

80

Fraction no. (b)

FIQ. 7. Analysis of early products of oalf thymus high molecular weight DNA polymeraseoatalyzed poly(dA) replication on a Sephadex 676 oolumn. The ma&ions were carried out for 20 min at 20°C. Each reaction mixture (0.26 ml) contains O-02 rd-KP, (pH 7*0), 1 mM-2-meraaptoethanol, 0.1 m&r-EDTA, 0.6 mm-Mm.&, 0.2 mrd-[nzeth$-3H]dTTP, 980 pmol d(pA)E, 1000 pmol l’4Cld(PT)~~,100 pg bovine serum albumin/ml, 36 units of enzyme, and 0 M, 0.06 M or 0.1 aa-NaCl. Analysis of the products of the reactions on a Sephadex G76 column was ae described in Materials and Methods.

3H-labeled monomer were used. The reaction was allowed to proceed to the extent of an average of one to two nucleotides added per initiator : template molecule. The degradative activities associated with E. wli polymerase I do not permit this hind of analysis, so only the mammalian enzymes were examined by this procedure. With these enzymes, all components and products of the reaction can be identified in the column fractions. The separation of the products of calf thymus high molecular weight DNA polymerase-catalyzed poly(dA) replication on a Sephadex G75 column is shown in Figure 7. The Figure illustrates the results obtained when the reactions were carried out at different ionic strengths. The average addition indicated in each panel of Figure 7 represents the extent of replication for the reaction analyzed. Each of these numbers was calculated from the ratio of the sum of 3H-labeled nucleotide to the sum of 14C-labeled oligonucleotide in the column fractions. The template chain elutes from the column in the void volume and is indicated in the Figure by the arrow. When the ionic strength of the reaction is low, 0.02 M-KP, (pH 7.0) in Figure 7(a), most of the product formed has more than one newly added nucleotide, showing a bias for the addition of several nucleotides to an initiator molecule prior to dissociation of the enzyme from that template : initiator complex. It should be noted, even in this least favorable case, that the amount of initiator bearing one or two additions exceeds the number of enzyme molecules present in the reaction, suggesting that the enzyme can dissooiate after a single addition. When the ionic strength of the polymerization

ENZYMATIC

DNA SYNTHESIS

231

60

70

80

Fraction no

90

100

110

120

130

140

150

160

170

180

190

200

210

220

Frochon no

FIG. 8. DEAE-cellulose column chromatography of calf thymus high molecular weight DNA polymerase products. The poly(dA) repliaation was carried out in a l-ml reaction containing 0.02 M-KP, (pH 7.0), 1 mm-2-meroaptoethanol, O-1 mM-EDTA, 0.6 m&r-MnCl,, 0.2 ma6-[methyl1OOpg bovine serum albumin/ml, 0.1 M-N&I 3H]dTTP, 3920 pmol d(pA)s, 4200 pmol [l*C]d(pT)I,, and 140 units of enzyme. Preliminary separation of the template and the product was carried out on a Sephadex G76 aolumn under alk~lme oonditions as described in Materials and Methods. Produot oligonuoleotides (fraction 66 to 82 of the Sephadex oolumn) were pooled, acidiiied and analyzed on a DEAE-oellulose column as described in Materials and Methods. The chromatograph shown in this Figure represents over 96% of the oligonucleotide fraction loaded onto the DE62 cellulose column.

reaction was increased by the addition of50 mM-NaCl (Fig. 7(b)), the size distribution of the product improves, and in the presence of 100 ma-NaCl (Fig. 7(c)) distribution of product indicates a nearly random interaction of the enzyme and template : initiator. The Sephadex 675 profiles of the size distribution of the products of poly(dA) replication do not produce a clear separation of the oligonucleotides. This separation can be improved by fractionation on DEAE-cellulose. Figure 8 shows the elution pattern of the products of calf thymus high molecular weight DNA polymerase from DE52 cellulose when the reaction was carried out in the presence of 100 mM-NaCl. In this experiment, the oligonucleotide products were first separated from the template by gel filtration on alkaline Sephadex 675 columns (insert in Fig. 8). The oligonucleotide fraction subject to analysis on DE52 cellulose represents over 90% of the radioactivity formed from monomer and 98% of the radioactivity associated with the initiator. Although the absolute amount of each oligonucleotide has not been estimated from the DE52 cellulose column fractions (Fig. 8) due to poor resolution, it is clear that the observed distribution approximates a Poisson distribution in which the average addition is about one (e.g. for x = 1, f = 0.37; x = 2, f = O-18; etc., where x = no. of additions and f = relative fkquency). The results from this

L. M. 8. CHANU

232

[NaCt] ~0.3 M Average addition

[NaCL]: 0.1 M Average addition 0.7

t

50

60

t

70 (a)

00

90

60

70

[NaCL] : 0.5 M Average addition 0.4

I.2

80

Fraction no. (b!

- 30 %

t

90

60

70

00

90

(c)

FIQ. 9. Analysis of early products of celf thymus low moleoular weight DNA polymerasec8t8lyzed poly(dA) replicetion on 8 Sephadex 676 oolumn. The reactions were carried out for 10 min at 20°C. Eech reaation contains O-06 M-Ammediol buffer (pH 84I), 0.6 mra-MnCl,, 1 mra-2mercaptoethanol, O-1 m&EDTA, 0.2 mx-[nz&hyZ-3H]dTTP, 100 pg bovine serum albumin/ml, 980 pmol d(pA);;-,, 1000 pmol d(pT)lS, 1.1 pmol enzyme, and 0.1 M, 0.3 Y or 0.6 M-Nacl. Analysis of the products of the reaotions on a Sephadex G76 oolumn w8s 8S desoribed in Materiala and Methods.

study show that when the high molecular weight DNA polymerase reaction is carried out at near physiological ionic strength, the interaction of the enzyme with the template is almost completely random, and translocation of chain on enzyme surface does not occur. The early products of calf thymus low molecular weight DNA polymerasecatalyzed poly(dA) replication in the presence of various amounts of NaCl were also analyzed on alkaline Sephadex G75 columns and the results are shown in Figure 9. The distribution obtained for the low molecular weight enzyme is similar to those obtained with the high molecular weight enzyme except that a much higher ionic strength is required to produce random interaction. Since the low molecular weight enzyme binds to denatured DNA cellulose eluting at about O-5 iv-NaCl and is a basic protein (Chang, 19733), these results are to be anticipated. The rate of the enzyme reaction is stimulated by the presence of 0.2 M-salt in the reaction (Chang, 1973o). At relatively low ionic strength (Fig. 9(a)), it is apparent that the enzyme tends to polymerize several nucleotides onto a template molecule prior to dissociation. Increasing the ionic strength of the reaction results in improved distribution of the product size (Fig. 9(b) and (c)). Even in the presence of O-5 ru-NaCl, the distribution of product shows bias toward multiple additions. However, under all three salt conditions tested, the number of product chains containing one addition exceeds the number of enzyme molecules in the reaction, suggesting that the enzyme can but does not necessarily dissociate from the template after a single addition.

ENZYMATIC

DNA

,Triphosphate

A

A

233

SYNTHESIS binding

Complex

A

I

sate

Complex

II

Complex

Reassociation

Fra. 10. Schematic products.

diagram

for the i&erections

of DNA

polymerase

with

substrates

and

It certainly can be argued that these secondary binding sites of the enzyme to the are part of the mechanism for translocating the chain on the enzyme surface during DNA synthesis. If this mechanism were to be solely responsible for translocation, it is rather ineffective since a generally distributive interaction is shown even at low ionic strength. It seems to me that it is more likely that the secondary binding sites on the enzyme surface may contribute to sequestration of the template near the active site of the enzyme molecule, but at the catalytic site of the enzyme, the growing end of the template probably dissociates from the enzyme after each polymerization reaction. Further reaction requires the reassociation of the growing end of the template at proper binding sites. A schematic diagram of the interactions between DNA polymerase, substrates and products is shown in Figure 10. The diagram includes only the interactions of the active site of the enzyme with the growing end of the template and does not consider secondary binding. Complex I depicts the growing end of the template and monomer at the proper binding sites on t’he enzyme surface, that is, the tertiary substrate : enzyme complex. Complex II shows the tertiary product : enzyme complex, formed by the nucleophilic attack of the 3’-hydroxyl group of the initiator chain at the cr-phosphoryl position of the deoxynucleoside triphosphate with formation of the phosphodiester bond and the release of pyrophosphate. At this stage two alternatives are possible; dissociation and translocation. The results presented indicate that instead of translocating the initiator : template complex on the enzyme surface to free the triphosphate binding site for further reaction, complex III is formed by the dissociation of the growing template

IU

234

L. M. S. CHANC

end of the template from the active site of the enzyme and reassociation of the template and the enzyme at proper binding sites again. If this model is correct then the enzyme-bound Zn atom postulated to be involved in the movement of the polymer chains on the enzymes surface (Slater et al., 1972) would appear likely to be involved only in the binding of the 3’-hydroxyl group of the growing chain (Chang & Bollum, 1970) and not in the translocation of the chains. If secondary binding sites for the polymer chains on the enzyme surface are available, it is possible that the template molecule will remain bound to the enzyme, but dissociation and reassociation of the growing end of the template still occurs, and other unbound template molecules could interact at the catalytic site. The secondary template binding sites do not seem to play a major role in governing the overall interactions between the enzyme and the template in the simple homogeneous systems examined since a generally distributive enzyme: template interaction can easily be demonstrated for all three DNA polymerases.

4. Discussion The results presented are clearly consistent with a distributive action of several DNA polymerase enzymes replicating homopolymer templates. Yet the replication of DNA ia vivo is sequential (or processive on a gross view) in that replication starts from a point on the chromosome and grows in a linear mamer. It would seem that a lot of time would be lost if the enzyme responsible for polymerization of nucleotides falls off the template after each addition of nucleotide at the 3’-terminus of the growing chain. The original statements are not incompatible, they are just not comparable. Enzymatic DNA synthesis may be classified as homogeneous catalysis, wherein all reactants and catalyst are free and diffusible in solution (or at least in one phase). DNA replication in vivo may be better classified as heterogeneous catalysis-where other phases may participate in an important way. The involvement of factors other than DNA polymerase in DNA synthesis in vivo (Alberts, 1973; Klein & Bonhoeffer, 1972) would be critical in the heterogeneous system. The possibility of membrane participation in DNA replication has also been suggested for bacterial systems (Sueoka & Quinn, 1968; Knippers & Stratling, 1970). It is quite likely that DNA polymerase is held near the growing point of the DNA by these additional factors during in vivo DNA replication. Such interactions between DNA polymerase, factors, and DNA would allow the enzyme dissociated from the growing point after a polymerization step to instantaneously reassociate at the proper binding sites. If the enzyme were held near the growing point in the manner proposed, the two important consequences would be an apparent movement of DNA on the enzyme surface and a much faster polymerization rate than could be predicted from the turnover number of the enzyme determined for the purified enzyme in homogeneous catalysis. This proposal is consistent with the arguments that isolated DNA polymerases have turnover numbers lower than that required to account for the rate of chain growth in DNA replication in vivo for both prokaryotic and eukaryotic systems (Cairns, 1963 ; Huberman & Riggs, 1968). By extension of the same logic, it is apparent that whether a DNA polymerase is involved in a heterogeneous replication process cannot be decided upon from its rate of homogeneous catalysis. Aside from the speculative aspects, the results presented indicate how one might approach the problem of DNA repliaation in systems not susceptible to genetic

ENZYMATIC

DNA

SYNTHESIS

236

oomplementation. Use of the method in Results, section (a) (which may be called “kinetic complementation”) with reconstructed systems might allow testing for conversion of a homogeneous catalysis to heterogeneous catalysis. A positive test requires a change to processive synthesis and a rate increase over the homogeneous system. Studies of this kind may also aid in establishing the roles of factors required for DNA synthesis in vivo in systems that are susceptible to genetic complementation. The discussion dealing with the heterogeneous catalysis that may occur in DNA replication in vivo does not imply a change in the molecular mechanisms of the enzyme

under heterogeneous

conditions.

Within

the DNA

replication

complex,

the

dissociation and reassociation of the enzyme with DNA must occur continuously. It is likely

that all deoxynucleotide-polymerizing

enzymes will operate in this manner

in homogeneous catalysis unless secondary factors perturb the system. The methods described can be used for the unambiguous analysis of mechanism in polymer template-directed

polymerizations.

This research was supported

by Public Health

Service Research Grant GM19789.

REFERENCES Alberta, B. (1973). In Molecular Cytogenetzcs (Hamkalo, B. A. & Papaconstantinou, J., eds), pp. 233-251, Plenum Publishing Corp., New York. Bollum, F. J. (1963). Cold Spring Harbor Symp. Quant. Bid. 28, 21-26. Bollum, F. J. (1904). S&me, 144, 560-661. Bollum, F. J. (1966). In Procedures in Nucleic Acid Research (Cantoni, G. 8: Davis, D., eds), pp. 677-682, Harper and Row, New York. Bollum, F. J. (1967). In Genetic Elements (Shugar, D., ed.), pp. 3-15, Academic Press, New York. Cairns, J. (1963). J. Mol. Biol. 6, 208-213. Chang, L. M. S. (1973a). J. Biol. Chem. 248, 6983-6992. Chang, L. M. S. (1973b). J. Biol. Chem. 248, 3789-3796. Chang, L. M. S. & Bollum, F. J. (1970). Proc. Nut. Ad. Sci., U.S.A. 65, 1041-1048. Chang, L. M. S. & Bollum, F. J. (1973). J. Biol. Chem. 248, 3398-3404. Chang, L. M. S., Cassani, G. R. & Bollum, F. J. (1972). J. Biol. Chem. 247, 7718-7723. Epstein, R. H., BoU, A., Steinberg, C. M., Kelkuberger, E., Boy De La Tour, E., Chevalky, R., Edgar, R. S., Susman, M., Denhardt, G. & Leilausis, A. (1963). Cold &wing Harbor Symp. Quant. Biol. 28, 375-394. Goulian, M. (1971). Annu. Rev. Biochem. 40, 855-898. Huberman, J. A. & Riggs, A. D. (1968). J. MOE. Biol. 32, 327-341. Klein, A. & Bonhoeffer, F. (1972). Annu. Rev. Biochem. 41, 301-332. Knippers, R. & Strathug, W. (1970). Nature (London), 226, 713-717. Koruberg, A. (1969). Science, 163, 1410-1418. Slater, J, P., Tamir, Z., Loeb, L. A. & Mildvan, A. S. (1972). J. Biol. Chem. 247,6784-6794. Sueoka, N. BE Quinn, W. G. (1968). Cold Spring Harbor Symp. Qumt. Biol. 33, 696-705. Yoneda, M. & Bolhun, F. J. (1965). J. Biol. Chem. 240, 3385-3391.