Polyacrylamide Gels - Science Direct

ANALYTICAL

BIOCHEMISTRY

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(1983)

Detection of Enzymatic Activities in Sodium Dodecyl SulfatePolyacrylamide Gels: DNA Polymerases as Model Enzymes’ A. BLANK, Department

JOHN R. SILBER, *y2 MICHAEL

of Biochemistry, Memorial

P. THELEN,

AND CHARLES

University of Call$ornia. Berkeley, California 94720. Cancer Research Laboratory, Department of Pathology University of Washington, Seattle, Washington 98195

and *The SM-30.

A. DEKKER~ Joseph

Gottstein

Received July 5, 1983 Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations. Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here. It was observed that reproducibility of a repotted procedure for DNA polymerasc detection (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hiibscher, U., and Banks, G. R. (1981) Nucl. Acids Rex 9, 1825- 1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al. A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described. The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis. These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase I and its Klenow fragment and nanogram amounts of calf thymus 01and rat liver (Novikoff hepatoma) p polymerases. Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities. KEY WORDS: DNA polymerases; enzymes (detection); proteins (renaturation); sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sodium dodecyl sulfate.

The activity of diverse enzymes (e.g. (1)) including DNA polymerases (2) has been detected in polyacrylamide gels following SDS4 eiectrophoresis. In contrast to protein staining methods, activity detection is based on enzymatic catalysis, and permits identification ’ This work was supported in part by grants from the National Institutes of Health (CA- 19606, CA-24845, and ES-01896). A preliminary account has appeared in abstract form (Blank, A., Silber, J. R., Thelen, M. P. and Dekker, C. A., Fed. Proc. 42, 2148 (1983)). ’ Present address: National Center for Toxicological Research, Jefferson, Arkansas 72079. 3 To whom correspondence should be addressed. 4 Abbreviations used: j3-ME, fl-mercaptoethanol; BSA, bovine serum albumin; NaPPi, sodium pyrophosphate; pol I, Escherichia coli polymerase I; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid, Tris, tris(hydroxymethyl)aminomethane. 423

and partial characterization of nanogram to picogram amounts of catalytically active species in crude cell and tissue homogenates. In spite of its advantages, activity detection in SDS gels is not widely utilized primarily because of the critical dependence of sensitivity on individual SDS preparations. This dependence has been documented in the case of RNases and DNases (3,4). Lack of reproducibility (5) associated with varying SDS preparations (6) has also been reported in the case of DNA polymerases. We show here that loss of sensitivity in DNA polymerase detection is caused, at least in part, by lipophilic contaminants in SDS (7) which inhibit renaturation. The loss can be mitigated by simple procedural modifications which presumably facilitate refolding of enzymes within the polyacrylamide 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved

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matrix. Moreover, the expedients which facilitate detection of DNA polymerases appear, by virtue of their mode of action, to be generally applicable to detection of catalytic activities in SDS-polyacrylamide gels.

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times with ether, and used without further treatment. The activated DNA was ~1.5% soluble in 3.5% perchloric acid at 0°C. Gels were run at 250 V until the dye marker entered the separating gel (20-25 min) and at 350 V until the dye was 0.5-l cm from the bottom EXPERIMENTAL PROCEDURES (about 50 min longer). Maximum current was ca. 80 mA. Occasionally gels were run at lower SDS used was BDH product 30176 (crys- voltage to give runs of 2-2.5 h without effect talline flakes, no longer available), Lot on the final autoradiogram. 20504 10; BDH product 442 15 (specially puBuffer A (0.01 M Tris-HCl, pH 7.5, 0.005 rified for biochemical work), Lot 2563030; M @-ME) containing various concentrations Bio-Rad electrophoresis grade, Lots 19760, of isopropanol was used to remove detergent 19632, and 18221-1. Purified pol I (22,000 from gels immediately following electrophounits/mg (8)) and Klenow fragment prepared resis. Rapid diffusion was maintained during therefrom (9) were gifts of Dr. L. A. Loeb. this step by gentle agitation. All solutions were Purified Novikoff /3 polymerase (58,100 units/ preequilibrated at the specified temperatures. mg (10)) was the gift of Dr. Dale Mosbaugh. After removal of detergent, gels were incubated Partially purified calf thymus LYpolymerase at 4°C for 19-25 h in 0.5 or 1 liter of 0.05 M (Lot 896-12, 90 units/&s0 unit) was from PTris-HCI, pH 7.5, 0.005 M ,&ME, 0.001 M L Biochemicals; 1 unit catalyzes the, incorEDTA to permit further renaturation (2). Viporation of 1 nmol of dAMP into an acidsualization of polymerase activity was precipitable product with poly dT * rA12-, 8 as achieved essentially as described (2,13). Gels template primer in 1 h at 37’C. A crude exwere incubated at 37°C for 19.5-21 h in ca. tract of Escherichiu coli (1 l), which was the 9 gel vol(lO0 ml/gel) of 0.07 M Tris-HCl, pH gift of Dr. R. M. Schaaper and Dr. L. A. Loeb, 7.5, 0.007 M MgC&, 0.01 M dithiothreitol, 14 contained 32 mg/ml of TCA-precipitable, PM each dATP, dGTP, and dCTP, and 0.8 Lowry-positive material when measured with &i/ml (ca. 1 nM) 32P-dTTP (New England a BSA standard (12). Nuclear, 600-800 Ci/mmol). Gels were then Electrophoresis was carried out essentially washed for 1 h at room temperature with genas described (2,13) using an apparatus in which tle stirring in four changes of 0.5 liter of 5% both faces of the gel sandwich are immersed TCA- 1% NaPPi ; they were held thereafter at in buffer. Separating gels (9 X 15 X 0.08 cm) 4°C in 0.5 liter of the same solution for 44contained 7.5% acrylamide (Bio-Rad), 0.2% 48 h with one change at about 24 h. Gels were bis-acrylamide (Bio-Rad), 0.1% SDS, 2.0 -4260 finally dried on Whatman 3MM paper and units/ml of activated DNA, 0.375 M Trisautoradiographed at -70°C using Kodak HCl, pH 8.8, 0.002 M EDTA, 0.063% am- XAR-2 film and a DuPont Cronex Lightening monium persulfate, and 0.05% (v/v) Plus intensifying screen, with exposure times N,N,N’,N’-tetramethylethylenediamine (Bio- as specified in figure legends. Individual secRad). Except where specified, separating gels tions of the same gel were realigned before contained 50 pg/ml of bovine fibrinogen drying, and were autoradiographed as a unit. (Sigma product F-4000). Stacking gels (4.5%) Autoradiograms were photographed under were 1 cm high. Calf thymus DNA was acstandardized conditions to preserve compativated by a modification of the procedure of rability. Spanos et al. (2). DNA (0.8 mg/ml) was inRESULTS cubated for 6 min at 37°C in 8 mM Tris-HCl, Figure 1 shows the effect of different SDS pH 8.0, 16 mM NaCl, 4 IIIM MgCl*, 0.8 mM preparations on detection of DNA polymerase EDTA, 200 &ml BSA, and 0.26 &ml activity by the method of Spanos and his colDNase I (Worthington). The reaction mixture was extracted twice with phenol and several leagues (2,13). Gel A was run with crystalline

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FIG. 1. DNA polymerase activity in gels run with different SDS preparations. Effects of embedding fibrinogen and ofremoving detergent with aqueous isopropanol. BDH crystalline SDS (discontinued product 30 176) was used for gel A,6 BDH contemporary SDS (product 442 15) for gel B. Gels A and B are otherwise essentially the same. Sections I and 2 of each gel were cast as a unit containing 50 &ml of fibrinogen in the matrix. Sections 3 and 4, which contained no fibrinogen, were likewise cast as a unit, separated from 1 and 2 by a polypropylene dividing strip. After electrophoresis, each of the units was cut in half to produce four sections per gel. To remove detergent, sections 1 and 4 were each incubated separately for 60 min at 37°C in 125 ml of buffer A containing 25% isopropanol (v/v); the solution was replaced every 20 min. To remove isopropanol, each section was then incubated for 30 min at 37°C in 125 ml of buffer A with one change after 15 min. Sections 2 and 3 were treated as above but isopropanol was omitted. The following schedule was maintained for further processing. Gel A: renaturation at 4°C 19 h; incubation with [32P]dTTP, 20.3 h; exposure to TCA solution at 4°C. 46 h; autoradiography, 44 h. Gel B: 19, 22.7, 48, and 46.5 h, respectively. Each well was loaded with a 10-J sample containing either 0.02 pl of Escherichiu coli extract, 0.65 ng rat liver B polymerase, or 0. I3 units of calf thymus (Ypolymerase.

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SDS from BDH (discontinued product 30 176), used exclusively by these authors.’ Gel B was run with SDS product 442 15 (“specially purified for biochemical work”) currently available from BDH. The particular lot used for gel B yields sensitivity representative of contemporary, electrophoresis grade SDS when tested in a standard procedure for activity staining RNases (4).6 Also shown are the effects of two simple modifications of the original procedure: embedding fibrinogen in the polyacrylamide matrix (sections 1 and 2) and removing detergent with aqueous isopropanol after electrophoresis (sections 1 and 4). Only in section 3 was the unmodified protocol utilized. Casting fibrinogen in gel matrices (section 2) increases sensitivity with both SDS preparations. However, activity observed with contemporary SDS (gel B) is not as great as with crystalline SDS (gel A). Washing gels with 25% isopropanol (section 4) also increases sensitivity, such that the two SDS preparations appear comparable. This result is analogous to that shown for RNases in gels 1A and 1B of Blank et al. (4). The combined effect of casting gels with fibrinogen and washing with isopropanol (section 1) depends on the SDS. With crystalline SDS, recovery of all polymerase activities in fibrinogen-cast gels is reduced by exposure to isopropanol. Of immediate practical significance however, are the results of gel B, run with a representative contemporary electrophoresis grade SDS, where washing with isopropanol uniformly improves ’ Crystalline SDS was used in gel preparation, in sample buffer, and in cathode buffer. To conserve this irreplaceable reagent, anode buffer was prepared with the same SDS used throughout gel B. The SDS in the anode buffer does not affect sensitivity (3,22) (see Discussion). 6 The commercial, electrophoresis grade SDS we have handled in our laboratory falls into three categories with respect to RNase detection (4). Superior: BDH product 30176, crystalline, no longer sold. See Fig. 1A of Ref. (4). Represenfutive: Encompasses all preparations we have handled except for the two specified under Superior and Poor, and includes eight listed in Ref. (4) and four BioRad preparations employed more recently. Results comparable to those in Fig. 1B of Ref. (4). Poor: Bio-Rad Lot 18221-l. See Fig. 1C of Ref. (4).

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recovery of activity. Thus, even though exposure of polymerases to 25% isopropanol is intrinsically damaging, as revealed in gel A, isopropanol treatment is necessary to counteract the deleterious effect of contemporary SDS. Indeed, sensitivity is enhanced over that observed using crystalline SDS in the original protocol (compare gel B, section 1 and gel A, section 3). As illustrated in Figs. 2 and 3, comparable sensitivity is obtained with other electrophoresis grade SDS preparations purchased from a different supplier. Figure 1 shows that the effect of isopropanol treatment depends on the SDS used and reflects a balance of beneficial and deleterious actions of the alcohol. It is therefore important to examine different conditions of exposure (concentration, duration, temperature) when studying any particular polymerase. Figure 2 shows the effect on calf thymus (Ypolymerase activity of varying the isopropanol concentration used to remove detergent. Treatment with isopropanol was for 45 min at 30°C. Best results were obtained at 20% (v/v); no activity at all was observed with buffer containing no

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FIG. 2. Effect of removing detergent in solutions of different isopropanol concentration. Wells in a single slab were loaded with 0.13 units of calf thymus a polymerase. After electrophoresis, the gel was cut into sections which were incubated individually in 100 ml of buffer A containing the specified concentrations (v/v) of isopropanol. Incubations were carried out at 30°C for 45 min using fresh solution every 15 min. Each section was then incubated for an additional 30 min in 100 ml of buffer A with one change after 15 min. The following schedule was maintained for further processing: renaturation at 4°C 23 h; incubation with [32P]dTTP, 19.5 h; exposure to TCA at 4°C 44 h; autoradiography, 120 h.

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FIG. 3. Sensitivity of detection. A single gel was loaded with (left to right): 445, 89, and 18 pg of pol I; 17, 3.4, and 0.67 pg of KIenow fragment; 9 and 1.7 ng of Novikoff hepatoma /3 polymerase; 0.13 and 0.026 units of calf thymus (Y polymerase. After electrophoresis, the gel was incubated for 60 min at room temperature in 0.5 liter of buffer A containing 20% isopropanol (v/v) with a change of solution every 15 min. The gel was incubated in 0.5 liter of buffer A for 30 min at room temperature with one change after 15 min. The following schedule was maintained thereafter: renaturation at 4°C 24 h; incubation with [3ZP]dTTP, 23.5 h; exposure to TCA solution at 4°C 46 h; autoradiography, 21.5 h (E. coli polymerases) or 68 h (01and fl polymerases).

isopropanol. It was also determined that 60 min exposure to 20% isopropanol at room temperature (25”Q rather than 30 or 37°C gave superior results (not shown). The SDS used for these experiments (Bio-Rad Lot 19760) is representative of the electrophoresis grade products we have handled (7). It should be noted that, because of intrinsic differences among enzymes, the above mentioned conditions may not be generally optimal. The results of Fig. 2 illustrate the sharp dependence of one commonly studied polymerase activity on the concentration of isopropanol used to remove detergent, and provide a guide for experimentation with other enzymes. We have estimated levels of detectability of several DNA polymerases in gels run with contemporary SDS. Figure 3 illustrates the sensitivity of detection of pol E and its Klenow fragment, of calf thymus a! polymerase and of Novikoff hepatoma p polymerase. The SDS used (Bio-Rad Lot 19632) is, again, a representative electrophoresis grade product (4,7).

Twenty picograms of pol I7 and less than 1 pg of Klenow fragment are readily detectable after 2 1 h of autoradiography; sensitivity can be increased, if required, by extended autoradiography. The polymerase activity of crude extracts of E. coli (20 nl containing < 650 ng of protein) is also easily visualized (Fig. 1B). Larger amounts of calf thymus (Yand Novikoff fl polymerases are required for visualization, than of pol I. The (Ybands in Fig. 3 are given by 0.13 and 0.026 units (estimated to be 110 and 22 ng, respectively (14,15)) of enzyme after 68 h of autoradiography. The @ bands, also visualized after 68 h, represent the activity of 9 and 1.7 ng, respectively, of enzyme. The amount of (Ypolymerase in the gels of Figs. ’ The pol I preparation, which had been stored 4 years at -7O”C, displays at least 7 catalytically active species, none of which comigrates with the apparently homogeneous Klenow fragment which had been similarly stored. Catalytically active, lower molecular weight polypeptides have been noted in purified pol 1 preparations and in E. coli extracts (23.13).

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1B (section l), 2, and 3 (lane 7) is the same, although each gel was run with a different SDS preparation. Inspection of these gels, with consideration of the varying times of autoradiography, reveals good reproducibility. To determine whether contemporary SDS contains impurities which interfere with activity detection of DNA polymerases, an electrophoresis grade preparation (Bio-Rad 1822 11) which has given exceptionally low recovery of RNase activity (Fig. lc of Blank et al. (4)), was extracted with petroleum ether. The lipophilic material obtained (4 mg/g of SDS) was added to electrophoresis buffer prepared with crystalline SDS (BDH 30 176). The effects on polymerase detection are seen in Fig. 4 which shows a gel loaded and run according to the protocol of Fig. 1. In comparison to the gel of Fig. lA, for which crystalline SDS with no added material was used, the gel of Fig. 4 shows a marked reduction of activity in sections 2-4 and approaches comparability with the gel of Fig. 1A only in section 1. Thus, the addition of 0.4% (w/w) of lipophilic impurities markedly diminishes sensitivity of detection. The deleterious effect is ameliorated by the combination of embedding fibrinogen in the gel and washing with isopropanol (section 1). To ascertain that the procedure used to extract lipophiles did not give rise to material which interferes with activity detection, a control gel was run in which an identically prepared extract of crystalline SDS (BDH 30 176) was substituted in an identical manner for the extract of Bio-Rad 1822 l- 1. When this extract of crystalline SDS was added to electrophoresis buffer containing crystalline SDS (BDH 30176), no diminution in activity was apparent relative to the gel of Fig. 1A (gel not shown). When Bio-Rad 1822 l-l SDS itself was substituted for crystalline SDS in the electrophoresis buffer of a gel which was loaded and run according to the protocol of Fig. 1, the activity detected was considerably less than that seen in Fig. 1B with a representative contemporary SDS preparation.‘j No activity was detected in sections 2-4, and only the pol I bands seen in Fig. 4 and a weak CYband were

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visible in section 1 (gel not shown). This severe impairment of DNA polymerase detection, which parallels severe impairment of RNase detection (Blank et al. (4)), is probably related to the high level of lipophilic contamination of Bio-Rad 1822 l- 1. The level is four times that of other, contemporary electrophoresis grade SDS preparations we have analyzed, including those used in this study (7). It should be noted that somewhat less activity was recovered when Bio-Rad 1822 I- 1 SDS was used instead of crystalline SDS compounded with lipophiles from Bio-Rad 18221- 1 as in Fig. 4. This observation suggests that extraction of lipophiles was not complete, and/or that addition of lipophiles to a solution of crystalline SDS does not produce mixed micelles identical to those formed by Bio-Rad 1822 1- 1 SDS itself ( 16). Nonlipophilic impurities -might also contribute to loss of sensitivity, although this SDS contains ~0.1% tetradecyl- and hexadecyl sulfates (4). DISCUSSION

Detection of enzymatic activity in SDS gels is an important recent technique which permits analysis of impure samples far too small to be subjected to classical methods of fractionation. The technique complements and extends traditional methods, having unique power to delineate the number, molecular weights, and isoelectric points of catalytically active species in diverse enzyme preparations (e.g., (17)). The technique requires renaturation of proteins, and several laboratories have experimented with alternative methods for achieving renaturation of enzymes and of binding proteins within the gel matrix (e.g., ( 1,18-20)). Dependence of renaturation on the SDS used, documented for RNases and DNases (3,4) may be a general phenomenon, seen here for DNA polymerases. Thus, the crystalline SDS used exclusively in the original work on detection of DNA polymerases, and no longer sold, is superior for detection of all three groups of enzymes. Moreover, the same contemporary SDS preparations which yield

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FIG. 4. Effect of lipophilic contaminants in SDS on detection of DNA polymerase activity. The gel and samples were prepared with BDH crystalline SDS, the cathode buffer with crystalline SDS to which the petroleum ether extractable material from Bio-Rad 18221-I SDS was added (see below) and the anode buffer with Bio-Rad 1822 1-1 SDS. Samples and protocol for removing detergent were the same as in Fig. 1. The schedule for further processing: renaturation at 4”C, 19 h; incubation in [32P]dTTP, 2 1 h; exposure to TCA solution at 4°C 48 h; autoradiography, 44 h. For lipophile extraction, SDS was ground to a uniformly fine powder and shaken gently overnight at room temperature in petroleum ether (Mallinckrodt reagent grade, boiling range 3%60°C 15 ml/g SDS). After centrifugation, the solvent was removed and the SDS was washed. briefly with further petroleum ether (5 ml/g SDS). The combined extracts were evaporated to dryness under a stream of N2. The extracted lipophiles were taken up in isopropanol (1 ml/ g SDS extracted) and added with vigorous stirring to a 10% solution (w/v) of crystalline SDS in H20; the total weight of crystalline SDS was the same as that weight of Bio-Rad 18221-I SDS from which the lipophiles had been extracted. The solution of compounded SDS was used to prepare cathode buffer.

reduced renaturation of RNases and DNases (4) give parallel reductions in the detection of DNA polymerases. The deleterious effect is probably due to lipophilic contaminants in the SDS which bind tightly to enzymes and impede renaturation. These lipophiles, which constitute about 0.1% by wt of most electrophoresis grade SDS preparations we have examined, include dodecyl alcohol, 1-dodecene, didodecyl ether, didodecyl sulfate, and long chain hydrocarbons (7). Like certain nonionic substances including dodecyl alcohol (2 1), they are transported from the cathode buffer into gels as components of negatively charged SDS

micelles. It is important to note that BDH crystalline SDS is free of detectable amounts of all of the above mentioned lipophiles except dodecyl alcohol which is the least inhibitory (7). We have employed two expedients to increase sensitivity and uniformity of DNA polymerase detection with contemporary electrophoresis grade SDS. One is to cast fibrinogen into the polyacrylamide matrix. Fibrinogen may promote renaturation by adsorbing inhibitory lipophiles from the electrophoresis buffer, thus sparing enzymes of interest, and by providing a more hydrophilic

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environment for refolding of enzymes. If these are among the mechanisms by which fibrinogen acts, inclusion of protein in gel matrices would be expected to promote renaturation of other enzymes as well. Indeed, casting of BSA in gels has been reported to improve detection of small amounts of some enzymes ( 1,4,1 7).899The second expedient we have employed is washing detergent from gels with aqueous isopropanol after electrophoresis. The organic solvent probably promotes renaturation by dissociating inhibitory lipophiles from enzyme molecules. The concomitant, deleterious effect of isopropanol observed for DNA polymerases is not universal, since isopropanol treatment does not adversely affect the RNases and DNases we have examined (4). In conclusion, it is important that our results in enhancing detection of DNA polymerases concur with findings concerning RNases and DNases. This concurrence indicates that the simple expedients described may facilitate detection of other enzymes and permit broad exploitation of a powerful technique. The concurrence also indicates that denaturation of enzymes by (pure) SDS may be a more generally reversible process than has been supposed. ACKNOWLEDGMENTS We thank Dr. Lawrence A. Loeb for his generous contribution of laboratory space and materials and for dis-

‘Fibrinogen was used because the smaller albumin molecules migrate out of the upper portion of gels during electrophoresis. 9 Inclusion of excess, heterogeneous protein in the ap plied sample, rather than in the gel, has improved detection of DNA polymerase activity (6). We have made a similar observation in the case of RNases. However, addition of a mixture of proteins to samples does not result in a uniform protein concentration in the gel. As a consequence, we observed selective renaturation of RNases at sites of high protein concentration, leading to distortion of isozyme profiles (Snook, J., Blank, A., and Dekker, C. A., unpublished data).

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cussions of the manuscript, Dr. Giovanna Ames for her valuable gift of BDH crystalline SDS, and Scientific Photography Laboratory for expert photography.

REFERENCES 1. Lacks, S. A., and Springhom, S. S. (1980) J. Biol. Chem. 255, 7461-1473. 2. Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hiibscher, U., and Banks, G. R. (198 1) Nucl. Acids Res. 9, 1825-1839. 3. Lacks, S. A., Springhorn, S. S., and Rosenthal, A. L. (1979) Anal. Biochem. 100, 357-363. 4. Blank, A., Sugiyama, R. H., and Dekker, C. A. ( 1982) Anal. Biochem. 120,267-275. 5. Filpula, D., Fisher, P. A., and Kom, D. (1982) J. Biol. Chem. 257, 2029-2040. 6. Karawya, E. M., and Wilson, S. H. (1982) J. Biol. Chem. 257, 13,129-13,134. 7. Thelen, M. P., Blank, A., McKeon, T. A., and Dekker, C. A. (1982) Fed. Proc. 41, 1203. 8. Jovin, T. M., Englund, P. T., and Bertsch, L. L. (1969) J. Biol. Chem. 244,2996-3008. 9. Klenow, H., and Overgaard-Hansen, K. (1970) Fed. Eur. Biochem. Sot. Lett. 6, 25-27. 10. Stalker, D. M., Mosbaugh, D. W., and Meyer, R. R. (1976) Biochemistry 15, 3114-3121. 11. Kombetg, A. ( 1974) DNA Synthesis, p. 2 17, Freeman, San Francisco. 12. Lowry, 0. H., Rosebrough, J. S., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275.

13. Hiibscher, U., Spanos, A., Albert, W., Grummt, F., and Banks, G. R. (198 1) Proc. Nat. Acad. Sci. USA 78,6771-6775. 14. Masaki, S., Koiwai, O., and Yoshida, S. (1982) J. Biol. Chem. 257,7172-7177. 15. Ma&i, S., and Yoshida, S. (1978) B&hem. Biophys. Acta 521, 74-88. 16. Menger, F. M. (1979) Act. Chem. Rex 12, 11 l-l 17. 17. Schieven, G. L., Blank, A., and Dekker, C. A. (1982) Biochemistry 21, 5148-5155. 18. Heussen, C., and Dowdle, E. B. ( 1980) Anal. B&hem. 102,

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19. Manrow, R. E., and Dottin, R. P. (1980) Proc. Nat. Acad. Sci. USA 17, 730-734. 20. Copeland, B. R., Richter, R. J., and Furlong, C. E. (1982) J. Biol. Chem. 257, 15,065-15,071. 21. Takagi, T., Kubo, K., Asakura, J., and Isemura, T. (1975) J. Biochem. (Tokyo) 78, 1297-1300. 22. Sugiyama, R. H. (1981) Ph.D. dissertation, University of California, Berkeley. 23. Yoshida, S., and Cavalieri, L. F. (1971) Proc. Nat. Acad. Sci. USA 68,200-204.