THE JOURNAL or BIOLOQICAL CHEUIST~Y Vol. 244, No. 11. Issue of June 10,pp. 3WW-3018, 1969 PrinluI in U.S.A. Enzymatic Synthesis of Deoxyribonucleic Acid XXVII. CHEMICAL MODIFICATIONS OF DEOXYRIBONUCLEIC ACID POLYMERASE* (Received for publication, November 6, 1968) THOMAS M. JOVIN,$ PAUL T. ENGLUND,~ AND ARTHUR KORNBERG From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 942 06 SUMMARY The purified Escherichia coli DNA polymerase contains a single sulfhydryl group which reacts with Hg(I1) to produce either a monomer contahing a single mercury atom or a dimer of two protein molecules joined by a mercury atom. Both forms retain full polymerase and exonuclease activities, im- plying that the sulfhydryl group is relatively exposed and not essential for enzymatic activity. The acylating agent, N-carboxymethylisatoic anhydride, reacts with an observed maximum of 11 sites on the enzyme to form a highly fluorescent derivative with altered functional properties. Only 0.2% of the original polymerase activity but 920% of the exonuclease activity are observed at pH 7.4 with DNA as primer or substrate. Measurements of release of inorganic pyrophosphate and binding of triphosphates indi- cate that the predominant effect of this acylating agent is to cause a marked reduction in the affinity for deoxyribonucleo- side triphosphate substrates, but concomitant changes in the interaction of the enzyme with polynucleotides are probably also involved. The data are discussed in terms of a model in which the several activities of the enzyme are catalyzed at the same active center, which is differentiated into physically distinct sites. In the preceding paper of this series (1) some of the physical and chemical characteristics of the purified Escherichia coli DNA polymerase were presented. This communicaticn will describe the effects of two chemical modifications on the physical and chemical properties of the enzyme. One of these, the addi- tion of a mercury atom to the single sulfhydryl group of the enzyme, does not affect any of its activities, and with the radio- * This study was supported in part by grants from the National Institutes of Health (United States Public Health Service), the National Science Foundation, and the National Aeronautics and Space Administration. 3 National Science Foundation Postdoctoral Fellow. Presently Established Investigator, American Heart Association; present address, Max Planck Institut fur physikalisehe Chemie, Got- tingen, Germany. $ National Institutes of Health Postdoctoral Fellow. Present address, Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. isotope, 20*Hg, provides an extremely sensitive tracer for physical studies. The other modification is the production of a fluores- cent derivative with the acylating agent, N-carboxymethylisatic anhydride. This derivative has useful properties for physical studies, and is the first example of a differential modification of the enzymatic activities associated with the DNA polymerase molecule. The virtual abolition of polymerase function with retention or even enhancement of the exonuclease activity, coupled with the indications that a single enzyme center encom- passes both functions, provides additional information about the active sites of the enzyme. EXPERlMENTAL PROCEDURE Materials 20sHg(N03)2 (in 2 N HNOa), obtained from New England Nuclear, was diluted to about 2 mM and neutralized with Tris. The Hg(I1) concentration was determined by a titration of re- duced glutathione based on the 5,5'-dithiobis(2-nitrobenzoic acid) sulfhydryl method of Ellman (2); 0.5 mole of Hg(I1) neutralized the reactivity of 1.0 mole of glutathione for 54'- dithiobis(2-nitrokenzoic acid). The specific radioactivity, deter- mined with a scintillation counter set for 14C counting, was 1.6 x 108 cpm pmole-1. The synthesis and properties of NCMIA: a gift of Dr. Lubert Stryer, will be described elsewhere.* 5,5'- Dithiobis(2-nitrobenzoic acid) was obtained commercially. DEAE paper was Whatman DE-81. Darco G-60 was a product of Atlas Chemical Industries. DNA polymerase was the homo- geneous Fraction 7; its concentration was based on a molecular weight of 109,000 (1). The sources of other materials and rea- gents are given in the previous paper (1). Methods Sucrose Gradient CentrifugationSucrose gradient centrifuga- tion was performed according to the method of Martin and Ames (3). Gradients (3.6 ml) of 5 to 20% sucrose in 0.2 M potassium phosphate buffer, pH 7.4, were centrifuged at 60,000 rpm in an International model B-60 centrifuge (SB 405 rotor) for 11 hours at 3'. Fractions were analyzed for enzymatic activity, radio- 1 The abbreviations used are: NCMIA, N-carboxymethylisatoic anhydride (N-carboxymethyl-N-carboxyanthranilic acid, cyclic anhydride); poly d(A-T), the alternating copolymer of deoxy- riboadenylate and deoxyribothymidylate previously designated dAT. * R. P. Haugland and L. Stryer, manuscript in preparation. 3009 3010 Enzymatic flyrunasis of DNA. XXVII Vol. 244, No. 11 None. . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . Glutathione, 0.02 M.. . . . . . . , . . . . . . . . . . dATP + dTTP, 0.05 mM each.. . . . . . . . Poly d(A-T), 0.1 mMa.. . . . . . . . . . . . . . . . Fraction FIG. 1. Gel filtration of polymerase allowed to react with excess *O3Hg(II). The 50-pl sample contained 1.8 nmoles of polymerase and 8.5 nmoles of '03Hg(II) in 0.1 M potassium phos- phate, pH 7.0. The protein had been incubated with the mercury for 15 min at 0". The column of Sephadex G-150 (74 cm X 0.07 cm2) was eluted with 0.2 M Tris-O.1 M HCI. Fractions of 0.19 ml were collected at a rate of 2.5 ml per hour at 4". The column was calibrated with blue dextran in a separate experiment. The polymerase and exonuclease assays were run without 2-mercapto- ethanol. Recovery of protein from the column was 62%. % % 0 2 87 98 8 4 4 3 I I n Fraction FIG. 2. Sucrose density gradient of *0SHg(II) allowed to react with excess polymerase. The 7O-pl sample contained 2.2 nmoles of polymerase and 1.06 nmoles of 20aHg(II) in 0.1 M potassium phosphate, pH 7.0. The protein had been incubated with the mercury for 43 hours at 0". Fractions of 0.14 ml were collected for radioactivity determinations, enzymatic assay, and protein determination. The sedimentation coefficients were determined by comparison with a hemoglobin marker in a separate tube. The polymerase and exonuclease assays were run without mer- captoethanol. The recovery of protein from the gradient was 82%. activity, and protein. Sedimentation coefficients were esti- mated with human hemoglobin as a marker (4.6 S) at concen- trations above 0.1 mg ml-l in order to preserve the tetrameric state (4). Fluorescence Spectra-Fluorescence emission and excitation spectra of NCMIA and its derivatives were made with an Aminco-Bowman spectrophotofluorometer equipped with an RCA 1P21 photomultiplier. The emission, excitation, and polarization measurements of the protein conjugates were per- formed with the recording spectrofluorometer described by Stryer (5). Microcuvette assemblies accommodating quartz tubes with an internal diameter of 3 mm were used. The emis- sion spectra are uncorrected for variations in sensitivity of the detection system with wavelength. Calibrations for the system have been given by Haugland and Stryer (6). Corrected flu- orescence excitation spectra and polarization measurements were recorded directly with the use of a ratio amplifier (5). In the case of polarization measurements, the exciting light was isolated with a Corning 7-31 filter. All measurements were made at 5" f 1". Other methods have already been described (I) or are given in the table and figure legends. Exonuclease assays were under conditions described for exonuclease I1 (l), except where other conditions are defined. RESULTS Reaction of DNA Polymerase with 208Hg(ZZ) With excess 20aHg(II), the enzyme, after isolation by gel filtra- tion, contained 1 mole of *NHg per mole of protein (Fig. I). The sedimentation coefficient of this derivative, as estimated by sucrose density gradient centrifugation, was identical with that of the untnodified enzyme (5.9 S), and agrees with the value of 5.6 S previously obtained by analytical centrifugation (1). TABLE I Stability of polymerase-mercury linkage The monomer and dimer were isolated by gel filtration and sucrose density centrifugation, respectively (Figs. 1 and 2). The 0.1-ml reaction mixtures, containing either 3.7 pmoles of monomer or 6.2 pmoles of dimer, in 0.05 M potassium phosphate, pH 7.2, were incubated for 10 min at 37". They were then precipitated with 0.2 ml of cold 10% trichloracetic acid and centrifuged for 10 min at 12,000 X g. The supernatant fluid (0.25 ml) was removed and neutralized with 0.7 ml of 0.8 M Tris. Mercury released from the protein was measured in a scintillation counter with the use of 10 ml of the dioxane fluid. 1 Mercury liberated from Treatment 1 Dimer 1 Monomer a In a separate experiment, 0.1 IMI poly d(A-T) was incubated with 1.08 p~ zOsHg(II). Any 2ozHg-poly d(A-T) complex formed must have been dissociated by the trichloracetic acid precipita- tion, because the mercury was recovered quantitatively in the supernatant fluid. Had mercury been removed by poly d(A-T) from the polymerase monomer or dimer, it would have appeared in the supernatant fluid aft,er the acid precipitation. Issue of June 10, 1969 T. M. Jovin, P. T. Englund, and A. Kornberg 3011 Since Hg(I1) is divalent, it might be expected to link polymerase molecules in dimers, as it does with serum albumin (7). This possibility was realized in a reaction mixture with a 2-fold excess of polymerase over Hg(I1) (Fig. 2). Peak I was desig- nated as dimer on the basis of a sedimentation coefficient of 7.9 S, compared to 5.9 S for the native enzyme, and the molar ratio of mercury to protein of 0.55. Peak ZI was a mixture of un- modified polymerase and mercury-polymerase which was still in the monomer state. Had the time of reaction been increased, all of the mercury might have been found in Peak I. In another experiment in which polymerase was incubated with mercury for only 20 min before the centrifugation, less than 25% of the *03Hg(II) wm incorporated into dimer. The specific activities of the mercury-containing monomer and dimer for both polymerase and exonuclease were the same as those of the unmodified enzyme in each experiment. It was necessary to establish, however, that components of the mixture used for assay of these activities, such as DNA, poly d(A-T), or triphosphates, did not remove the mercury and regenerate the 0. 0 h .- i " 3 V - 0. ? 0 0 0 IIII! FIG. 3. Absorption spectra of NCMIA and derivatives. a, NCMIA inO.1 M potassium phosphate, pH 7.4; b, reaction product of NCMIA in 0.1 M potassium phosphate, pH 7.4, after 23 hours at 37" (no further spectral change was observed after 15 additional hours); c, reaction product of NCMIA after ammonolysis in 13.3 M NHa for 21 hours at room temperature, in 0.1 M potassium phos- phate, pH 7.4. All solutions were 0.1 mM by reference to an original NCMIA solution prepared by weight with the use of a molecular weight of 239 (this value includes 1 water molecule of crystallization). The spectra were recorded on a Cary 14 spec- trophotometer at 4-10". The absorption maxima, in millimicrons, of the three compounds were a, 243 and 317; b, 2-51 and 331; and e, 253 and 334. FIQ. 4. Fluorescence emission spectra of NCMIA and deriva- tives. Spectra a, b, and c are of the compounds described in the legend to Fig. 3. Relative quantum yields were calculated by dividing the peak emission intensity by the absorbance at 330 mp. The emission maxima, in milli- microns, of the three compounds were a, 395; b, 412; and c, 438. Excitation was at 330 mp. native enzvme. Englund, Kelly, and Kornberg have shown elsewhere that mercury is not removed by DNA or poly d(A-T) (8). Furthermore, the results in Table I also indicate that the mercury-polymerase linkage is stable in the presence of poly d(A-T) and triphosphates, but that the mercury is quantitatively removed by glutathione. Since care was taken to omit a sulfhy- dryl reagent from the assay solution and from the enzyme diluent, it is likely that the mercury-polymerase linkage was intact during the assay. Jovin, Englund, and Bertsch have previously shown that carboxymethylation of the single sulfhydryl group also does not affect either enzymatic activity (1). Electroqhoresis of mercury-Folvmerasq pya ra t,imm? fin- ~$-y:- acrylamide gels showed that in Buffer System A (I), the mono- meric form migrated with the same mobility as the unmodified enzyme. However, a mercury-polymerase preparation in which the formation of dimers was favored showed, in 9% polyacryl- amide gels, a new zone with a mobility of 0.75 relative to that of the more prominent monomer zone. The presence of a diffuse zone of protein between the zones suggested that the equilibrium between the two states was under constant readjustment during the electrophoretic separation, possibly because of dissociation and counter migration of Hg(I1). Reactions of DNA Polymerase with NCMIA Properties of NCMZA-The absorption and fluorescence emission spectra of NCMIA (I) and some derivatives are shown in Figs. 3 and 4, respectively. Hydrolysis at 37" in 0.1 M potassium phosphate, pH 7.4, caused a 9-fold increase in quantum yield and red shifts of 14 mp in the absorption spectrum and 17 mp in the emission spectrum. The half-life in the same buffer at 0" was about 30 hours when measured by changes in absorbance at 260 mp, in contrast with the reported half-life of 13 min for isatoic anhydride at 25" (9). The observation of time-dependent isos- bestic and isoemissive points during the course of the reaction at 0" indicated the presence of more than two species and suggested that a sequence of hydrolytic reactions was involved. Ammonolysis of NCMIA also caused an 8-fold increase in quantum yield and red shifts of 17 mp in the absorption and 43 mp in the emission spectra (Figs. 3 and 4). NCMIA in 13.3 M NHa had a half-life of 2 hours at approximately 20" and 1.15 hours at 28". This relative stability is surprising inview of the re- 3012 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11 TABLE I1 Properties of DNA polymerase derivatives prepared with NCMIA Property Preparation" NCMIA (mM). ...... dATP (mM). ................................ Polymerase (mM). .................................................. Moles dye per mole enzymeb. ....................................... NCMIA reacted (%). .............................................. Stoichiometry of conjugation Absorption spectra A980: Atao. .......................................................... A332:A2so ........................................................... Fluorescence spectra E4,7:E336d .......................................................... Energy transfer (%)e.. ............................................ Emission anisotropy!. .............................................. Quantum yield of conjugated dyer. ................................. Enzymatic activitiesh Polymerase (poly d(A-T) primer) .............. Polymerase (DNA primer). .................... .......... .......... tio DNA primer to poly d(A-T) primer. .............. ..................................................... a The incubations, in 40 pl, were for 40 hours at 0'. The buffer waa 0.05 M potassium phosphate, pH 7.5. After reaction, the solutions were diluted 6-fold and dialyzed extensively against the same buffer. The spectra were recorded without further dilu- tions. *Calculated from difference spectra (eg. Fig. 5) using an mp value of 8 X 1oP r1 cm-' for the conjugated dye (Fig. 3c). Owing to many uncertainties, the true values might differ by about 30%. c These values are subject to potentially large error from light scattering. d Ratio of emission intensities at the indicated wave lengths (millimicrons), with excitation at 290 mp. e Calculated from the excitation spectra (e.g. Fig. 6B) as de- scribed in the text. 1 The emission anisotropy, A, is given by (Fz - F,)/(F. + W,), where F, and Fu are the emission intensities with the analy- zer set parallel and perpendicular to the polarizer, respectively. r Quantum yield of fluorescence emission is defined as the ratio of emitted to absorbed quanta. An emission spectrum in which energy transfer from tryptophan to a conjugated dye is observed can be used to calculate quantum yield, Q, according to the formula Q = K (Edyo/Etrp) (1 - T)/T, where Edrs and Et,, are 0 bH,-COO- I I1 1 0.50 0.043 6.6 55 0.98 0.26 1.77 0.166 0.6 47 0.006 0.42 Derivative 2 0.47 0.70 0.039 6.1 51 1.02 0.25 1.65 0.166 0.5 43 0.013 0.08 6 0.51 3 0.089 0.044 2.3 100 1.40 0.13= 0.47 0.155 0.4 0.10 0.27 2.7 1.18 24 4 0.077 0.72 0.040 2.2 92 1.44 0.13~ 0.41 0.155 0.4 0.30 0.45 1.5 1.10 20 the intensities of the two peaks in the spectrum representing emis- sion by the dye (due to excitation by energy transfer) and the pro- tein emission (directly excited), respectively; Tis the efficiency of energy transfer, measured from an excitation spectrum; and K is an instrument constant (generally wave length-dependent). The term Etrp T/(1 - 2') is a measure of the exciting energy because it represents the change in the tryptophan emission intensity due to energy transfer. The value of K for the particu- lar instrument used in this study was calculated by use of the above equation with the data for anthraniloylchymotrypsin (Fig. 1 of Haugland and Stryer (6)), a system in which Q was measured directly. This value of K (0.29) was then used to calculate the quantum yields for the polymerase-NCMIA deriva- tives using the data given in this table. Because the anthra- niloylchymotrypsin and NCMIA-polymerase spectra had peaks with similar half-widths and maxima (within 5 mp), peak fluores- cence intensities rather than the integrated emission curves could be used. Expressed as ratios relative to unmodified polymerase. Exonuclease I11 was not used in the polymerase assays (1). iThe scale is normalized so that the value for unmodified polymerase is 1. port that isatoic anhydride and N-methylisatoic anhydride (10,ll) react within 5 min under similar conditions. Assuming the same reaction pathway as observed with N-methylktoic anhydride (10, ll), the most probable product of the ammonolysis of NCMIA is N-carboxylmethylanthranilamide (11). Fluorescence excitation spectra of the parent compound and the products of its reaction with water and ammonia were com- patible with the absorption spectra, although the peaks were shiftedta longer wave lengths by an average of 8 mp, presumably because of a varying emission intensity in this region of the spectrum. Spectral Properties of NCMIA-DNA Polymerase Conjqdes- Issue of June 10,1969 T. M. Jowin, P. T. England, and A. Kornberg 3013 DNA polymerase was allowed to react with NCMIA at neutral pH and 0". The reisolated protein had spectral properties consistent with the covalent attachment of the dye. In one experiment, polymerase was incubated with two levels of NCMIA in the presence and in the absence of dATP (Table 11). The absorption spectra of two of these derivatives are shown in Fig. 5. The dserence absorption spectra had peaks at 254 and 334 mp, and were similar to the spectra for the reaction product of NCMIA and ammonia (Fig. 3). The degree of conjugation was a function of the input concentration of NCMIA and of the time of incubation; the kinetics of conjugation will be described below. The number of dye molecules per protein molecule was calculated by using a molar extinction coefficient for the conjugated NCMIA of 8 x los M+ cm-1 at 254 mp (Fig. 3c). These values and other data for the derivatives are given in Table 11. The extent of reaction of NCMIA at the lower concentration was 90 to 100%; at the higher concentration it was 50 to 60%. As a test for the covalent nature of the attachment, the products were subjected to electrophoresis on a 9% polyacrylamide gel with Buffer System A. Fluorescent zones were detected which corresponded with the positions of the protein zones. Derivatives 3 and 4 showed somewhat skewed distributions favoring the less mobile protein; the center of mass had a mobility about 5% greater than that of the control enzyme. Derivative 2 had a broader, more symmetrical protein distribution, the center of which migrated about 16% faster than unmodified polymerase. This qualitative effect was expected, since each conjugated dye molecule contrib- utes at least one negative charge in the form of a carboxyl group IIIIIIII B 13 - E, 306 02 240 280 320 360 240 280 320 363 400 Wavelength (mA) !.5 P 13 ; 3 3.1 FIG. 5. Absorption spectra of DNA polymerase allowed to react with NCMIA. A, Derivative 2; B, Derivative 4. The preparation of both derivatives is described in Table 11. The solid lines are the absorption spectra of the dye-protein con- jugate. A*m is the absorbance measured with a 1-cm light path. The curve8 formed from individual points are difference spectra for the conjugated dye obtained by pointwise subtraction of the unmodified polymerase spectrum from that of each conjugate. All spectra were normalized to a protein concentration of 1.09 mg ml-' (1W6 M) by first estimating the conjugation stoichiometry from the absorbances at 280 and 260 mp, applying a correction factor to for the dye contribution (less than 3%), and ad- justing the spectra to a corrected Asso of 0.926 (actual absorb- ances were within 14y0 of this value). Protein concentrations measured by the method of Lowry et al. (12) gave values within experimental error of those determined spectrophotometrically. The data were recorded on a Zeiss PMQ I1 spectrophotometer with 1-cm light path cuvettes at a temperature of 2-10', Correc- tions for light scattering were not applied. 523 360 400 440 480 I:IIIIIII B Wavelength (my) FIG. 6. Fluorescence emission spectra (A) and excitation spectra (B) of DNA polymerase allowed to react with NCMIA. The numbers I and 4 refer to the corresponding derivatives of Table I1 and the number 0 to unreacted polymerase. Excitation for the emission spectra was at 290 mp. In the case of the excita- tionspectra, Corning CS 3-73 filters were used to isolate the emission. and, if conjugated with an amino group of lysine, results in the removal of a fractional positive charge. We calculated that a DNA polymerase molecule has 50 to 70 negative charges under the conditions of electrophoresis (pH 9.8 at O"), based on its amino acid composition, the assumption that the ammonia is derived from amide groups, and the reported values for the in- trinsic dissociation constants of the amino acid side groups (13). An incxease of mobility of 16% for Derivative 2 would therefore correspond to a change of about 8 to 11 negative charges as compared with the conjugation stoichiometry of 6. Fluorescence emission spectra of Derivatives 2 and 4 are shown in Fig. 6A; corresponding data for the other derivatives are given in Table 11. Native DNA polymerase had a trypto- phan emission at 335 mp when excited at 290 mp. Reaction with NCMIA reduced the 335 mp emission, and a new peak appeared at 427 mp. The effect was more pronounced in the case of the more highly conjugated derivatives. The emission spectrum of the conjugated dye, excited directly at 365 mp, also had a peak at 427 mp and thus resembled the spectrum of the presumed amide derivative of NCMIA (Fig. 3). Together with the similarity in the absorption spectra, this finding suggests that a large fraction of the dye may have been conjugated with amino groups on the protein. If so, the probable structure would be the N-crtrboxyanthranilamide (11), with R the side group of lysine. The single nonessential sulfhydryl group and NH2- terminal methionine (I), if involved in the reaction with NCMIA, cannot account stoichiometrically for the extent of reaction. The excitation spectra of Derivatives '2 and 4 (Fig. 6B) con- firmed that the tryptophan emission was quenched because of energy transfer to the dye chromophore. The three peaks in the spectra were in good correspondence with the absorption peaks (Fig. 5). The efficiency of energy transfer, T, was obtained 3014 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11 from the escitakion spectra by the following relation (6) T = n' (E¶SO/E334 - cd4 c~~~/& where E280 and EW are the magnitudes of the excitation spectra at 280 and 334 mp, respectively; 6280 and €334 are the molar extinc- tion coefficients of the conjugated dye at 280 and 334 mp with values of 360 and 3480 M? cm-1 (Fig. 3), respectively; is the molar extinction coefficient of the protein at 280 mp and is equal to 9.26 x 104 M-' cm-'; and n is the number of dye molecules per protein molecule. The calculated efficiencies of energy transfer ranged from 20 to 47% (Table 11). Since these values were necessarily averages for mixed populations of different molecules, quantitative deductions as to the probable situation of dye molecules relative to the nine tryptophan residues in the enzyme are not justified. The polarization of the fluorescence emission of the conjugated dye was measured; values are given in Table I1 in the form of emission anisotropies. The excited state lifetime of the con- jugated chromophore is not yet known; therefore an evaluation of the rotational relaxation time was not attempted. The quantum yield of the conjugated dye was not measured directly; estimated values of 0.4 to 0.6 were obtained by an indirect method (Table 11). Enzymatic Proper'ies of NCMIA-DNA Polymerase Conjugates -NCMIA had pronounced effects upon the enzymatic proper- ties of DNA polymerase, most notably a marked depression of polymerase activity (Table 11). The specific activity of Deriva- tive 1, having about 6.6 dye molecules per protein molecule, was reduced by more than 99% (poly d(A-TI-primed assay). Deriv- ative 3, with only about 2.3 dye molecules per protein molecule, showed 10% residual activity. When measured in the DNA- primed polymerase assay, the apparent reduction of activity was less (Table 11). The derivatives formed in the presence of dATP retained somewhat more polymerase activity despite only a slight reduction in conjugation! The presence of Mg++, which has been found recently to facilitate binding of deoxy- nucleoside triphosphates (14), did not make the conjugation less pronounced in preliminary experiments. The effects on exonuclease activity produced by reaction with NCMIA were strikingly different from those on polymerase activity. Low levels of conjugation (Derivatives 3 and 4, Table 11) actually led to an increase in specific activity of 10 to 20%, whereas higher degrees of reaction (Derivatives 1 and 2) depressed the activity but only by 20 to 50%. The relatively greater destruction of the polymerase activity compared to exonuclease activity suggested the possibility that a modification of the enzyme in the site for triphosphate binding and reactions might be responsible for this effect. To test this possibility, an NCMIA derivative of the enzyme was prepared in which polymerase activity was reduced to 0.02% in a poly d(A-T)-primed assay (Table 111). The exonuclease activity was 20% of that of the unmodified enzyme when tested with a poly d(A-T) substrate under the usual conditions of assay but 58% at pH 6.5 and 920% with a DNA substrate at pH 7.4. With this derivative, the functions involving triphos- 3 In another experiment, performed in parallel with that of Table 111, an NCMIA derivative of the enzyme was formed in the presence of a saturating concentration of poly d(A-T) oligomer (8). This enzyme had 25 times more polymerase activity and 3 times more exonuclease activity than enzyme modified in the absence of poly d(A-T) oligomer. The conjugation stoichiometry of this derivative was not measured. phates, such as PP, exchange, stimulation of exonuclease activity, and binding, were either markedly depressed or unde- tectable. However, pyrophosphorolysis, in which triphosphates appear as products, was catalyzed at the relatively high level of 2%. If the triphosphate site was altered in the reaction with NCMIA, an increase in K, for triphosphate in the synthetic reaction might be anticipated. In order to differentiate between the rates of phosphodiester bond formation and net synthesis, simultaneous measurements of both nucleotide incorporation into product and PPi release were made as a function of triphos- phate concentration (Fig. 7). The unmodified enzyme showed a typical Michaelis-Menten substrate saturation curve with the same kinetic parameters for the two functions (K, of 8 PM and vmax of 24,000 units mg-1). The NCMIA derivative showed a strikingly different behavior, with a clear dissociation between synthesis and PPi release. Whereas synthesis remained at a constant and low level, the rate of PPi release increased linearly with substrate concentration, suggesting that the K, for triphos- phates was in fact much greater. These results are all consistent with a profound distortion of the triphosphate site in the modified enzyme. In another experiment, the specific activity of synthesis was shown to be independent of enzyme concentration over a 25-fold range and was equivalent to the incorporation of only about two nucleotide units at the end of the polynucleotide chain bound to a given polymerase molecule. This calculation assumes the existence of a uniform population of enzyme molecules. An alternative possibility is that all the residual synthetic activity resided in a small fraction of relatively unaltered enzyme and that the PP, release represented a catalytic function aborted before actual phosphodiester bond formation. The postulate of a limited synthesis by most or all molecules, however, is consis- tent with the high level of residual nuclease activity. It would appear that the rate of synthesis is so reduced that the product is rapidly degraded leaving only a small steady state amount at the end of the chain. There is effectively, therefore, a turnover of substrate without a net prolongation. Assuming the validity of the proposed model, the linear dependence of PPi release on substrate concentration in the case of the modified enzyme would be consistent with a much in- creased K, and thus conditions of limiting substrate for which u = S.u,,/K,. The slope of 2.6 X 10' therefore represents the ratio of the altered kinetic parameters, um.,/K,. If pyro- phosphorolysis is the reversal of phosphodiester bond formation, then thermodynamic considerations stipulate that the ratio of these activities should not change in the modified enzyme when measured at equilibrium or even under the nonequilibrium assay conditions, assuming additionally that the catalytic mechanisms are not altered, and that the assay for the reverse reaction con- forms to the same limiting rate law, as stated above. Although these conditions have not been established, it is instructive to consider their possible validity in the light of the experimental results. The ratio of the specific activities for synthesis (or PPi release) and pyrophosphorolysis for the unmodified enzyme is 14.9 from Table 111. The modified enzyme showed 24 units mg-1 for pyrophosphorolysis, which would correspond to a u,,, of 360 units mg-l for PPi release assuming the invariance of the ratio of activities. It follows from the observed value of vmax/Km that K, should be on the order of 2 mM. In other experiments, Issue of June 10, 1969 T. M. Jowin, P. T. Enylund, and A. Kornberg 3015 18,000b 4,800 450 1,500 2,680 4,740 6,640 6,370 5,460 3,G00 5,020 13 58 1 , 210 6,050 -5,400 TABLE 111 Altered enzymatic activities of an NCMIA derivative of polymerase The NCMIA derivative was prepared in a reaction mixture phosphorolysis and PP, exchange were measured according to the containing 22 ALM enzyme, 30 PM mHg(II), and 1.6 mM NCMIA, method of Deutscher and Kornberg (X), except that the PPI in 0.1 M potassium phosphate, pH 7.4. After 25 hours at 0" the concentration was 1.3 mM. PPI exchange rates were calculated solution was extensively dialyzed against the same buffer. The assuming that, for the unmodified enzyme, pyrophosphorolysis is isolated material had a protein concentration of 1.93 mg ml-l both 50% inhibited by triphosphates under the conditions used (15) by the method of Lowry et al., and by corrected spectral deter- but that for the modified enzyme the activities are additive. minations. The absorbance ratio, A~so:A2oo, was 0.794 and the The binding of dTTP to the enzyme was measured by equilibrium number of conjugated dye molecules per protein molecule was dialysis at 5" in 0.05 M potassium phosphate, pH 7.4-7 mM MgCIL; calculated to be 10.7 by the method described in Table 11. The the method will be described elsewhere (14). For primer or mercury was present in the incubation in order to provide a substrate, 'H-DNA from E. coli was prepared according to the convenient determinant of concentration. It had no apparent procedure of Deutscher and Kornberg (15); the specific activity effect on the course of the reaction or on the properties of the was 1,140 cpm nmole-I. Single strand breaks were introduced derivative, although this point was not systematically in- by incubation with pancreatic DNase (Worthington) at a level of vestigated. All assays were performed at 37" for 30 min under 0.1 pg per pmole of DNA for 100 min at 37" before addition of conditions for which activity was proportional to enzyme con- polymerase and triphosphates. In the case of the poly d(A-T) as centration. One unit of activity corresponds to the incorpora- primer, the triphosphates dATP and dTTPwere addedat the indi- tion, release, or exchange of 10 nmoles of nucleotide per 30 min. cated concentrations. With DNA as primer or substrate, dGTP The polymerase and nuclease assays were carried out as pre- and dCTP were also supplied. Except where indicated, pH 3.2 12 260 260 320 360 1,170 1,410 1,440 940 990 120 120 24 25 -1 viously described (l), except for the indicated changes. Activity Polymerase Exonuclease Pyrophosp horolysis Pyrophosphorolysis + PPi exchange PPi exchange Binding of dTTP Primer or substrate Poly d(A-T) DNA (activated, calf thy- mus) Poly d(A-T) DNA (activated E. co2i) Poly d(A-T) Poly d(A-T) Poly d(A-T) rro- values were at 2.5'. Tri- bosphate PY 33 33 2 333d 33 33 Pi refers to potassium phosphate, Tris to Tris-HCI, and Gly to sodium glycinate. The concentration in all assays was 67 mM. * Exonuclease 111 was not required with this particular poly d(A-T) primer for the full expression of polymerase activity (1). saturation of the PPi release function was not achieved at a 0.8 mM triphosphate concentration, a result consistent with the predicted K,. Exteni of Conjagation of Enzpne-We wished to determine the minimum number of dye molecules required for alterations of enzymatic activity and to assess possible specificity for sites on the protein. The maximum stoichiometry of conjugation ob- served was about 11 dye molecules per protein molecule (Table 111). One simple model postulates the existence of N equally reactive sites, of which m are important for enzymatic function, so that reaction at any one or more virtually abolishes activity. The fraction, f, of residual activity in a population with an aver- age of p dye molecules per protein molecule would then be given 7.4 7.4 6.5@ 6.98' 7.325- 7. 90c 7. 6gC 8.16O 8.%= 8.65~ 8.76c~ 7.4 7.4 7.4 7.4 7.4 7.4 Buffer' Pi Pi Pi Pi Pi Pi Tris Tris GlY GlY G~Y Pi Pi Pi Pi Pi Pi I Modified enzyme Residual activity % 0.018 0.25 58 17 12 8 18 22 26 26 20 920 210 2.0 0.41 -0.018 < 10 c pH at 37". d The activation by triphosphates of the exonuclease function the unmodified enzyme is described by Lehman (16). by (1 - p/N)". Assuming N = 11, then a p of 2.3 andf of 0.1 (Table 11) lead to a value for m of about 10. The data for Deriv- ative 1 of Table I1 yield a value form of 6. KimtiCs of Conjugation of Enzyme-This model was evaluated further by studying the kinetics of reaction with DNA polym- erase at several concentrations of NCMIA (Fig. 8). Depres- sion of polymerase function proceeded from zero time with neither a lag nor a transient activation (Fig. 8A). This finding is consistent with "one-hit" kinetics, an effect dependent on the reaction of a single NCMIA molecule. The time course of exonuclease activity (Fig. 8B) was quite different from that of the polymerase. At the highest NCMIA concentration, the activity at the first time point (0.4 hour) was 3016 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11 U /-UnrnodiF,hd enzyme .e, 20,000 >r sv) 3t <5 10,000 .- + 8 u 0. [jH-dTTP) or [3'P-T-dATP] GYM) FIG. 7. Dependence of synthesis and PPi release kinetics on substrate concentration for unmodified and NCMIA-conjugated enzyme. The derivative described in Table I11 was used. Syn- thesis and PP, release were measured concurrently in a reaction 1 volume of 0.6 ml containing 67 mM potassium phosphate, pH 7.4, 6.7 mM MgCL, 1 mM mercaptoethanol, 31 nmoles of poly d(A-T), the indicated concentrations of "-dTTP (specific activity, 3.7 x 10' cpm nmole-1) and yS2P-dATP (specific activity, 7.7 x 10' cpm nmole-I), and either 4 ng of unmodified enzyme, 1.5 pg of the modified enzyme, or polymerase diluent alone. The poly d(A-T) was incubated for 30 rnin at 37" prior to the addition of triphos- phates and enzyme with 0.1 unit of exonuclease I11 per nmole of polynucleotide in order to increase its priming efficiency (1); the exonuclease I11 was inactivated by heating for 5 min at 65". After all additions, incubation was resumed for 30 min. The degree of incorporation of 3H-dTME' into polymer was measured by a modification of the procedure of Falaschi, Adler, and Xho- rana (17). A 0.1-ml aliquot of each incubation mixture was plated on a 2-cm square of DEAE-cellulose paper and dropped into 500 ml of 0.3 M ammonium formate (brought to pH 8 with NITa). The papers were shaken occasionally for 10 min at room temperature and drained. This process was repeated four times, followed by two washes each with 50 ml of ethanol and ether. The air dried papers were counted in 5 ml of a standard toluene scintillation fluid. The blanks were linearly related to the input of triphosphate, and the proportionality factor was 0.03%. PPi release was measured by a combination of adsorption to charcoal to remove nonutiliaed dATP and precipitation of interfering azP, from the supernatant by formation of the insoluble triethyl- ammonium-phosphomolybdate complex according to the method of Sugino and Miyoshi (18). To the remainder of the chilled incubation mixture (0.5 ml) were added 1 ml of 3 mM sodium pyrophosphate-0.15 N HCl and 6 drops (0.15 ml) of a 20% sus- pension of Darco G-60. After 5 rnin at 0" the suspension was centrifuged and 0.5 ml of the supernatant fluid was placed in a polyethylene scintillation vial with 0.05 ml of 2 M Tris base and 10 ml of the naphthalene-dioxane scintillation fluid. Blanks at this step were proportional to substrate input, with a factor of 1.6%. Controls indicated that the Darco was capable of ad- sorbing 99.6% of a pure triphosphate. Thus about 1% of the Y-SzP-dATP appeared to have been degraded during the incu- bation at 37". The 12Pi so produced was precipitated by treat- ment of an additional 0.8 ml of the Darco supernatant with 0.1 ml of 4 M HClO,, 0.7 ml of 0.08 M ammonium molybdate, and 0.14 ml of 0.8 M triethylammonium-HC1 (pH 5). The dense, yellow precipitate was removed by centrifugation and 1.3 ml of the supernatant containing the 3lPPi were placed in a polyethylene scintillation vial with 0.2 ml of 2 M Tris base and 10 ml of scintilla- tion fluid. This procedure reduced the blank to 0.5% of the input triphosphate, thus permitting this experiment with the modified enzyme when the maximal utilization of substrate was only 0.7%. The data for the unmodified enzyme were analyzed by plotting the observed incorporation, AS, against -In (1 - AS/S,), where So is the initial concentration of each triphosphate. This inte- grated form of the rate equation was used in order to correct for A Polymerase NCMIA A 0.2 3 I I 1 I i I 1 I 20 40 60 80 0 Tima in houi-5 FIG. 8. Kinetics of NCMIA reaction with DNA polymerase. A, Polymerase activity (poly d(A-T)-primed); B, exonuclease activity. Incubation waa at 0" in volumes of 12 pl containing 0.98 mg of polymerase ml-1 in 0.1 M potassium phosphate buffer, pH 7.1, and the indicated concentrations of NCMIA. The values are relative to unmodified polymerase (set at 1.0). Aliquots of 2 pl were removed at the indicated times for assay. actually above the control level and remained so even at 5.5 hours, when 84% of the polymerase activity had been lost. Only uFon further reaction was a decrease in exonuclease function observed. The limiting value of 48% to which exonuclease activity decreased agreed within experimental error with the values for Derivatives 1 and 2 (Table 11). At the intermediate NCMIA concentration, only enhancement of exonuclease activity was observed and the time course was also compatible with a single hit process. The maximum observed increase was about 30% (at 24 hours), with some indication of an incipient decline at about 72 hours. In order to determine rate constants, the logarithm of the the 1 to 8% utilization of substrate in the 30-min incubation period. A straight line was obtained for both PPI release and synthesis, yielding a K,,, of 8.1 p~ and a urn.= in the form of a specific activity of 24,000 units mg-1. The rate corresponding to the usual substrate concentration of 33 p~ was 19,300, within experi- mental error of usually observed values. Issue of June 10, 1969 T. M. Jovin, P. T. Englund, and A. Kornberg 3017 residual polymerase activity in Fig. 8 was plotted against a time axis corrected for NCMIA hydrolysis. Reasonably straight lines were obtained; they yielded an apparent second order rate constant of 5 M? min-1. Assuming a value for m of 10, the rate constant expressed with respect to sites instead of molecules would be 10 times less, or 0.5 M-1 min-1. The limited data do not permit a more detailed analysis, but it is of interest that the apparent second order rate constant for the reaction of NCMIA with NHa (20", 13.3 M NHa) was 3.6 x 10-4 b1-1 min-', or three orders of magnitude less.4 The incubation mixtures of Fig. 8 were analyzed electropho- retically 2 weeks after the last indicated time point. The three proteins, in order of increasing initial NCMIA concentration, migrated faster than unmodified polymerase by factors of 3%, 5%, and 23%. By comparison with the data for Derivative 2 (Table 11), it was calculated that the conjugates (Fig. 8) formed at 0.1 and 1 m~ NCMIA, respectively, had an average of about 2 and 8 dye molecules per polymerase molecule. Thus the initial increase in exonuclease activity observed in Fig. 8B occurred under conditions where on the average less than one NCMIA molecule had reacted and suggests that attachment of a single molecule affects both polymerase and exonuclease activi- ties concurrently. From the data of Fig. 8 it was estimated that the quantitative effect of a single substitution is to increase the nuclease function of the enzyme by a factor of about 2. Subse- quent reaction reduces activity, and thus in a mixed population the full potentiation is not seen. DISCUSSION The reactions of DNA polymerase with Hg(I1) confirm the observation from alkylation experiments that the single sulfhy- dryl group of the enzyme is not required for activity (1). The formation of a polymerase dimer bridged by a mercury atom indicates that the cysteine side chains are located in a relatively exposed site on the surface of the molecule. It is of interest that both members of the dimer retain full enzymatic activity. The possibility of forming dimeric structures through a direct disul- fide bond, as is possible with serum albumin (19), was not ex- plored. If such dimers or related forms exist in the cell, they would not have been detected because of the use of reducing agents in our purification procedure. DNA polymerase exhibits a quite different behavior in its reaction with the fluorescent acylating agent, N-carboxymethyl- isatoic anhydride. A product of limited acylation containing an average of 2.3 moles of dye per mole of protein showed a 20% increase in exonuclease, but its polymerase activity was reduced by 90%. In a more extensive reaction in which approximately 11 dye molecules were conjugated to the enzyme, only 0.02% of the polymerase activity remained while 20% or more of the exonuclease activity was preserved with poly d(A-T) as a primer or substrate. These are the first instances of selective inactiva- tion of the polymerase function, excluding the effects seen after treatment with guanidine-HCl and mercaptoethanol and de- scribed in the preceding paper (1). Prior studies have shown the constancy of the polymerase to nuclease ratio throughout purification and during heat and urea inactivation (20). These 4 This calculation does not take into account the probable nature of the reactive species in concentrated ammonia solutions. In reactions of isatoic anhydride and N-methylisatoic anhydride with n-butylamine at 1.3", rate constants of 180 M? sec-1 and 86 ~-1 sec-1, respectively, have been reported (9). and related observations (8, 14, 15) suggest, therefore, that the two catalytic functions are carried out in the same general active center of the molecule but that the sites do not correspond exactly. Inactivation of an enzyme by a modifying agent may often involve an alteration rather than abolition of function, as through shifts in pH maxima and relative affinities to substrates and cofactors. This point is supported by studies relating the vari- ous activities of polymerization, hydrolysis, pyrophosphate exchange, and pyrophosphorolysis. The finding of approxi- mately 11 reactive sites is of interest because there are potentially a much greater number of suitable groups on the protein, e.g. 61 c-amino groups of lysine. It is possible that sites of unusual reactivity are involved: in this regard the much lower reaction rate of NCMIA with ammonia is noteworthy. The functional properties of the modified enzyme and direct binding measurements indicate a large reduction in the affinity of the enzyme for the deoxyribonucleoside triphosphates. How- ever, other sites and effects must be involved. The protection from inactivation by poly d(A-T) suggests some capacity to redirect the course of reaction but probably not through direct interaction with NCMIA, since isatoic anhydride has been reported to be nonreactive toward phosphate and phosphate esters, including nucleotides (21). It will be of interest to deter- mine the effect of structure of various nucleic acids and nucleo- tides on this protective function. The altered pattern of nuclease function also indicates involve- ment of sites other than that for the triphosphates. Shifts in the pH spectrum of activity were observed, along with indica- tions of altered influences of ionic strength. More remarkable was the fact that at pH 7.4 the extensively conjugated enzyme was almost 10 times more effective with a DNA substrate but 10 times less effective with poly d(A-T) as compared with the original exonuclease, although in terms of absolute rates the poly d(A-T) was still the better substrate. Such an effect may reflect subtle changes in the influence of polynucleotide secondary structure upon the interaction with the enzyme. To what extent the exonuclease measurements reflect the act.ivity on the 3' terminus as compared with the activity directed toward the 5' terminus (22) has not been determined. In view of the recent evidence that the regions in the active center of the enzyme responsible for these two exonuclease activities of DNA polymerase are distinctly different,' it becomes of great interest to analyze the modified enzyme for each of these activities. The kinetic model proposed for the inactivation of the enzyme is not unique, owing to the probable existence of sites with vary- ing reactivity and functional importance. Clarification, of this problem requires the preparation and isolation, as by ion ex- change chromatography, of molecules with known and homoge- neous extents of conjugation. In order to refine the spectral studies, additional information about NCMIA and its derivatives is required. Model compounds would be of help in the evalua- tion of the nature of the substitution and the polarity of the modified site. Better quantitative determination of conjugation stoichiometry would follow from the use of radioactive reagent. It remains to be seen whether energy transfer, quantum yield, and polarization measurements are sensitive to the interaction of the enzyme with DNA and substrates. Finally, similar 6 N. R. Cozzarelli, R. B. Kelly, and A. Kornberg, manuscript in preparation. 3018 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11 studiea with other polymerases and reagents should indicate whether the phenomenon is a general one or specific to NCMIA and the E. coli enzyme. REFERENCES 1. JOVIN, T. M., ENGLUND, P. T., AND BERTSCH, L. L., J. Biol. 2. ELLMAN, G. L., Arch. Biochem. Biophys., 82, 70 (1959). 3. MARTIN, R. G., AND AMES, B. N., J. Biol. Chem., 2S6, 1372 4. SCHACHMAN. H. K.. AND EDELSTEIN. S. J.. Biochemistrv. 6. Chem., 244, 2996 (1969). (1961). "I . 2681 (1966). 5. STRYER. L., J. Mol. Biol.. 13. 482 (1965). 6. HAUGLAND,' R. P., AND STRYER, L.; in 6. N. RAMACHANDRAN (Editor), Conformation of biopolymers, Vol. I, Academic Press, New York, 1967, p. 231. 7. HUGHES, W. L., JR., J. Amer. Chem. Soc., 69, 1836 (1947). 8. ENGLUND, P. T., KELLY, R. B., AND KORNBERG, A., J. Biol. 9. BUNNETT, J. F., AND NAFF, M. B., J. Amer. Chem. SOC., 88, Chem., 244, 3045 (1969). 4001 (1966). 10. STAIGER, R. P., AND WAGNER, E. C., J. Org. Chem., 13, 347 11. STAIGER, R. P., AND MILLER, E. B., J. Org. Chem., 24, 1214 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, 13. EDSALL, J. T., AND WYMAN, J., Biophysical chemistry, Vol. I, 14. ENGLUND, P. T., HUBERMAN, J. A., JOYIN, T. M., AND KORN- 15. DEUTSCHER, M. P., AND KORNBEBG, A., J. Biol. Chem., 244, 16. LEHMAN, I. R., Annu. Reo. Biochem., 36, 645 (1967). 17. FALASCHI, A., ADLER, J., AND KHORANA, H. G., J. Biol. Chem., 18. SUGINO, Y., AND MIYOSHI, Y., J. Biol. Chem., 2S9,2360 (1964). 19. STRAESSLE, R., J. Amer. Chem. Soc., 76, 3138 (1954). 20. RICHARDSON, C. C., SCHILDKRAUT, C. L., APOSHIAN, H. V., AND KORNBERG, A., J. Biol. Chem., 239, 222 (1964). 21. STAIGER, R. P., AND WAGNER, E. C., J. Org. Chem., 18. 1427 (1953). 22. DEUTSCHER, M. P., AND KORNBERG, A., J. Biol. Chem., 244, 3029 (1969). (1948). (1959). R. J., J. Biol. Chem., 193, '26.5 (1951). Academic Press, New York, 1958, p. 534. BERG, A., J. Biol. Chem., 244, 3038 (1969). 3019 (1969). 238. 3080 (1963).