Proc. Nadl. Acad. Sci. USAVol. 81, pp. 7777-7781, December 1984Biochemistry

Identification of a higher molecular weight DNA polymerase acatalytic polypeptide in monkey cells by monoclonal antibody

(mammalian DNA polymerase a/renaturation of enzyme)

ESSAM KARAWYA*, JUDITH SWACK*, WALTRAUD ALBERT*t, JOSEPH FEDORKOt, JOHN D. MINNAt,AND SAMUEL H. WILSON**Laboratory of Biochemistry and WNational Cancer Institute-Navy Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD20205; and tInstitut fur Biochemie, Universitat Wurzburg, Wurzburg, Federal Republic of Germany

Communicated by Maxine Singer, August 28, 1984

ABSTRACT A monoclonal antibody against purified calfDNA polymerase a (deoxynucleosidetriphosphate:DNA deoxy-nucleotidyltransferase, EC 2.7.7.7) was used to immunopre-cipitate proteins from a crude soluble extract of growing mon-key BSC-1 cells. Immunoprecipitates contained familiar DNApolymerase a catalytic polypeptides of Mrs 115,000 and70,000 and also a M, 40,000 catalytic polypeptide; the majorcomponent in the immunoprecipitates, however, was a poly-peptide of Mr 190,000 not previously identified as a DNApolymerase. This protein was capable of DNA polymerase ac-tivity after electroelution from NaDodSO4/polyacrylamidegels and renaturation. The highly purified enzyme so obtainedwas active with poly(dT)-oligo(rA) as template-primer, resist-ant to dideoxy TTP (ddTTP), and inhibited by aphidicolin andbutylphenyldeoxyguanosine 5'-triphosphate, thus identifyingit as a DNA polymerase a. The results indicate that a polypep-tide of Mr 190,000 is an abundant component among DNApolymerase a catalytic polypeptides in growing monkey cells.

Most attempts to elucidate the subunit structure of DNApolymerase a (a-polymerase; deoxynucleosidetriphos-phate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) haverelied upon NaDodSO4/polyacrylamide gel electrophoreticanalysis of highly purified preparations. However, identifi-cation of enzyme subunits by this approach is complicatedby the possibilities of both proteolytic degradation and elimi-nation of important enzyme polypeptides during laboriousisolation procedures. Clearly, additional methods, not in-volving laborious purification, are needed for the study of a-polymerase polypeptides. Recently, "activity gel" analysis(1-3) of crude homogenates resulted in the identification of aMr 110,000-120,000 a-polymerase catalytic polypeptide in anumber of eukaryotic tissues; in addition, putative a-poly-merase catalytic polypeptides also were detected at aboutMr 70,000 (1-3). Nevertheless, use of the activity gel methodis complicated by the fact that the detection efficiency of a-polymerases is low, and some purified a-polymerases arecompletely unable to produce an activity signal (2). A prom-ising immunological approach, based upon solid-phase im-munobinding, was recently reported by Sauer and Lehman(4). Those workers observed a Mr 182,000 polypeptide incrude extracts ofDrosophila embryo that cross-reacted withan antiserum raised against the Mr 148,000 polypeptide ofpurified Drosophila a-polymerase; generally, a-polymerasesubunits of Mr> 160,000 had not been reported prior to thiswork. Lehman and co-workers (5, 6) subsequently showedthat the Mr 182,000 polypeptide itself was capable of DNApolymerase catalytic activity.

Studies of a-polymerases have been facilitated recently bythe development of monoclonal antibodies to these en-

zymes. Kom and co-workers (7, 8) used a monoclonal anti-body to KB cell a-polymerase to localize the enzyme by im-munocytofluorescence, and this group and Wahl et al. useda monoclonal antibody to purify the enzyme by immunoaf-finity chromatography (9, 10). Masaki et al. (11) found that amonoclonal antibody to calf a-polymerase could distinguishindividual species of the enzyme in partially purified prepa-rations from calf thymus, and Matsukage et al. (12) used amonoclonal antibody to chicken a-polymerase to study tis-sue-specific expression of the enzyme as a function of em-bryonic development. In the present study, we used a mono-clonal antibody approach to elucidate components of mam-malian cell lines that share immunological determinants withpurified a-polymerase. Proteins in a crude soluble extractfrom growth-phase monkey cells were subjected to immuno-precipitation with one of our monoclonal antibodies to a-polymerase. Immunoprecipitated polypeptides were electro-phoresed in NaDodSO4/polyacrylamide gels and then exam-ined for DNA polymerase activity. The results of thesestudies indicate that a crude soluble extract of growing mon-key cells contains a relatively abundant a-polymerase cata-lytic polypeptide of Mr 190,000. Several other polypep-tides were immunoprecipitated from the crude extract also,including familiar a-polymerase constituents of Mr 110,000-120,000 (1, 2, 13), Mr 64,000-70,000 (3, 14), and Mr 51,000(13-20).

MATERIALS AND METHODSEmbryonic bovine trachea (EBT) cells from the AmericanType Culture Collection, and African green monkey kidney(BSC-1) cells were maintained as described (2). Commercialpreparations of partially purified calf thymus a-polymeraseand near-homogeneous Escherichia coli DNA polymerase Ilarge fragment were from Worthington and New EnglandNuclear, respectively. DNA polymerase P was purified fromcalf thymus essentially as described by Tanabe et al. (21).Gelatin type IV from calf skin and Nonidet P-40 were fromSigma. [3 S]Methionine (specific activity, 1200 Ci/mmol; 1Ci = 37 GBq) and deoxythymidine [a-32P]triphosphate werefrom Amersham. Rabbit anti-rat 1gM serum was from Miles.The total IgG fraction was purified by chromatography onDEAE Affi-Gel blue and protein A-Sepharose. Componentsfor DNA polymerase assays were as described (13). 14C-la-beled marker proteins were from New England Nuclear.

Labeling of Cells with [35S]Methionine. Cells grown toabout midlogarithmic phase in 150-mm Petri dishes werewashed three times with phosphate-buffered saline (Pi/NaCl). The cell monolayer then was incubated for 6 hr at37°C in 5 ml of methionine-free Dulbecco-Vogt-modifiedEagle's medium containing 10% dialyzed fetal calf serum;

Abbreviations: a-polymerase, DNA polymerase a; EBT cells, em-bryonic bovine trachea cells; MC pol 1 and 2, monoclonal antibodiesto calf a-polymerase; ddTTP, dideoxy TTP.

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7778 Biochemistry: Karawya et aL.

this solution also contained 1 mCi Of [35S]methionine perdish.

Preparation of Cell Extracts. The culture fluid was re-moved, and the cell monolayers were washed three timeswith cold P1/NaCl and harvested by scraping. Cell pelletswere homogenized in -3 vol of 20 mM Tris HCI, pH 7.8/0.1mM EDTA/4 mM magnesium acetate/0.25 M sucrose/i mMdithiothreitol by using sonic disruption as described (22).The homogenates were centrifuged at 18,000 x g for 15 minat 40C, and the supernatant fractions were centrifuged againat 30,000 x g for 30 mmn at 40C. The clear supernatant frac-tions were used immediately for immunoprecipitation orkept frozen at -70'C; thawed extracts were centrifuged at40,000 x g for 20 min before use.

Immunoprecipitation. DNA polymerases in crude extractswere immunoprecipitated with a double-antibody immuno-precipitation method. The mixture for antigen-first antibodycomplex formation (186 ul) contained 0.16% gelatin, 0.8%Nonidet P-40, 15 mM NaCl, 0.05 mM EDTA, 0.5 mM dithio-threitol, 2 mM magnesium acetate, 0.125 M sucrose, 15 mMTris HCl (pH 7.8), 150,g ofIgM (either immune ornoimm-mune) per ml, and 120,ul of cell extract. This mixture wasincubated at4°C for 4 hr. Then 197,ul of a P1/NaCl solutioncontaining 890 ug of rabbit IgG fraction against ratIgM wasadded, and incubation at4°C was continued for 12 hr. Thesolution was centrifuged at 18,000 x g for 20min. The pelletfraction was resuspended four times at25°C in 0.1% Na-DodSO4/0.5% Nonidet P-40 in Pi/NaCl with centrifugationat15°C and 18,000 x g for 20min. The final washed pelletfraction was dissolved in 1.2% NaDodSO4/0.6% 2-mercap-toethanol/11% glycerol and heat-treated for 3min at100°Cbefore application to NaDodSO4/polyacrylamide gels.NaDodSO4/Polyacrylamide Slab Gel Electrophoresis and

Activity Gel Analysis. NaDodSO4/polyacrylamide gel elec-trophoresis was performed essentially as described (23). Foractivity gel analysis, we modified (24) a previously describedprotocol to increase the sensitivity of detection of a-poly-merase catalytic polypeptides. In summary, after the elec-trophoretic run in a gel containing activated calf thymusDNA, the gel was soaked first at room temperature in 1 literof 50 mM Tris-HCl (pH 7.6) for 15 min with one bufferchange and next at4°C for 3 hr in 1 liter of a buffer contain-ing 50 mM Tris HCl (pH 7.6), 6 mM magnesium acetate, 40mMKCl, 1 mM dithiothreitol, 0.01 mM EDTA, 16% glycer-ol, and 400 mg of bovine serum albumin. The gel was thenincubated at37°C for 24 hr in 40 ml of the same buffer con-taining 12.5,uM each of dATP, dCTP, and dGTP; 1,uMdTTP; and 0.25mCiof [32P]dTTP. The unincorporated ra-dioactivity was removed by washing the gel with 5% tri-chloroacetic acid/1% sodium pyrophosphate (2).

Autoradiography. The NaDodSO4/polyacrylamide gels,loaded with [35S]methionine-labeled samples, were soakedfor 1 hr in dimethyl sulfoxide followed by 2 hr in a solution of20% 2,5-diphenyloxazole in dimethyl sulfoxide, and finallywere soaked in distilled water for 1 hr with several changes.The gels were dried on 3 MM paper and exposed to KodakXAR-5 film with a Dupont Lightning Plus screen at -70°Cfor the desired period of time.

Electroelution and Renaturation of DNA Polymerase Poly-peptides. Electrophoresis was in a routine NaDodSO4/10%polyacrylamide slab gel (23) 100 mm in length. Molecularweights were assigned from marker proteins run in parallellanes that were cut from the gel and stained with Coomassieblue. Marker proteins were myosin, 8-galactosidase, phos-phorylase a, bovine serum albumin, and ovalbumin; migra-tions were 15.7, 27.9, 32.5, 47.7, and 68.6 mm, respectively.Portions of the gel containing polypeptides of interest wereexcised by slicing. Slices corresponding to peptide bands ofMrs 190,000, 115,000, 70,000, and 40,000 were 2-3 mm wide.Slices could be stored at 40C for 16 hr. Polypeptides were

electroeluted at 250C by using an ISCO model 1750 concen-trator. Elution was for 2 hr at 3 W with 25 mM Tris/195 mMglycine/0.1% NaDodSO4. The solution of eluted proteins(200 Al) was mixed with an equal volume of buffer D (50 mMTris HCI, pH 7.8/0.1 mM EDTA/10 mM 2-mercapto-ethanol/6 M urea), and the mixture at 250C was adjusted to 6M by addition of solid urea (25). The solution was adjusted tocontain bovine serum albumin at5 mg/ml and applied to a 1-ml column of Dowex 1-X8 equilibrated in buffer D at 250C.The flow-through in a volume of 500Al was mixed with anequal volume of 50 mM Tris HCI, pH 7.8/0.1 mM EDTA/10mM 2-mercaptoethanol/5O mM KCI/20% glycerol at 40C.The mixture was dialyzed against this same buffer for 4 hr at40C. The resulting solution of '800,ul was mixed with 0.5vol of 100% glycerol and stored at -200C. Renatured a-poly-merase polypeptides retained about 25% of the original ac-tivity after storage for 1 month; these enzymes lost activityupon freezing and thawing.

RESULTSImmunoprecipitation with 35S-Labeled Cells. Previously

we isolated mouse-rat hybridoma cell lines secreting ratIgM antibodies that reacted with homogeneous calf a-poly-merase in radioimmunoassay (unpublished data); one ofthese antibodies, termed monoclonal polymerase 1 (MC pol1), was used in the work to be described. In immunoprecip-itation experiments, this antibody was able to remove a-polymerase activity from the BSC-1 cell crude extract usedhere (unpublished data). These cells in growth phase wereexposed to medium containing [35S]methionine for 6 hr; a1.5Fa

a

0.75 -

0 1

[

! I_Top~~~~~~ ~~~~~lSO.11 10 73 66 51 44

ur.imun31 Dye

FrontTop 190 115 106 73 66 51 42 40

MD x 10 3

FIG. 1. Autoradiograms and densitometric tracings of a Na-DodSO4/8% polyacrylamide slab gel after electrophoresis of[35S]methionine-labeled proteins immunoprecipitated with MC pol1. Growing BSC-1 cells were labeled with [35S]methionine for 6 hr.Extraction of soluble proteins and immunoprecipitation were as de-scribed; a portion of the immunoprecipitate containing 186,000 dpmwas analyzed. The protein markers and Rf values were: myosin,0.29; /3-galactosidase, 0.35; phosphorylase a, 0.42; bovine serumalbumin, 0.6; and ovalbumin, 0.76. The Rf value for the main labeledband was 0.295. Exposure in a was for 1 hr and in b was for 3 hr.

1677A1551

Proc. NatL Acad ScL USA 81 (1984)

Proc. NatL Acad Scl USA 81 (1984) 7779

soluble extract was prepared, and antibody-antigen com-plexes eventually were immunoprecipitated with a secondantibody against rat IgM. Immunoprecipitates were washedby repeated resuspension at 250C in 0.5% Nonidet P-40/0.1% NaDodSO4 and then subjected to NaDodSO4/po-lyacrylamide gel electrophoresis. A typical pattern of im-munoprecipitated 35S-labeled polypeptides is shown in Fig.la. One prominent band at Mr = 190,000, migrating -1 mmahead of the myosin marker, was observed in the 8% gelalong with weaker bands at Mrs of 40,000 and 42,000. Densi-tometer tracing indicated that the ratio of label in the poly-peptides of Mrs 190,000 and 40,000 was about 6:1. Longerexposure revealed that several other polypeptides had beenimmunoprecipitated also (Fig. lb). Immunoprecipitates pre-pared with nonimmune rat IgM in this experiment failed toshow a significant amount of labeled protein in the gel (notshown).

In our experience, some variation among experiments wasobserved in the pattern of immunoprecipitated polypeptides(not shown). The Mr 42,000 band was not present in someexperiments, and in other experiments it was stronger thanin Fig. 1. Cells at higher confluency generally yielded pat-terns with less of the Mr 190,000 band. However, we consis-tently found that the polypeptide of Mr 190,000 was a ma-jor component of the immunoprecipitates. It is noted thatonly very minor bands were detected in the molecular weightrange of -125,000 to 175,000, which corresponds to the high-er molecular weight polypeptides reported in a number ofpreparations of purified a-polymerase.

Electroelution and Renaturation of ImmunoprecipitatedPolypeptides. Immunoprecipitates were prepared with MCpol 1 and an extract from unlabeled BSC-1 cells. After rou-tine electrophoresis in a NaDodSO4/10% polyacrylamidegel, individual polypeptides and regions of the gel corre-

sponding to Mr = 40,000-190,000 were removed from theunstained gel by slicing. Proteins in each gel slice were elec-troeluted and then separated from NaDodSO4 by chromatog-raphy over a small column of Dowex-1 in the presence of 6M urea (25). Urea was removed by dialysis, and the resultingsolutions of renatured proteins were assayed for DNA poly-merase activity (Table 1). Activity was observed with eachsample of renatured polypeptide; however, somewhat moreactivity was observed with the polypeptides of Mrs 190,000and 40,000 than with the other immunoprecipitated polypep-tides. No activity was detected with immunoprecipitatesprepared with nonimmune rat IgM.Recovery of activity of reference DNA polymerases in a

typical experiment (Table 1) was 8%, 3%, and 7%, respec-tively, for the partially purified preparation of calf a-poly-merase, purified calf 3-polymerase, and E. coli DNA poly-merase I large fragment. These values were relative to theactivities of reference enzymes at the beginning of the ex-periment. However, recoveries based upon activities of ref-erence enzymes that were mixed with urea and dialyzed inparallel with the electroeluted samples were about 10-foldhigher than the values shown in Table 1. It is noted that ac-tivity of the partially purified a-polymerase preparation wasdistributed in six regions of the gel corresponding to Mrsfrom 4190,000 to =40,000.Examination of some catalytic properties of the renatured

immunoprecipitated Mr 190,000 polypeptide revealed thatthe enzyme was active with poly(dT)-oligo(rA) as template-primer, as expected of an a-polymerase (Table 2). The poly-merization rate with activated DNA was not inhibited morethan =50% by ddTTP but was inhibited strongly by aphidi-colin and butylphenyldeoxyguanosine 5'-triphosphate. As il-lustrated in Table 2, these catalytic properties are consistentwith an a-polymerase but not with aB-polymerase. It should

Table 1. DNA polymerase activity of renatured proteins after electroelution from NaDodSO4/polyacrylamide gels

Protein electrophoresedImmunoprecipitates of BSC-1 cell extractt

Gel regionelectroeluted,M X 10o-3

>190190 ± 9180-120115 ± 4110-7570 ± 465-4340 ± 2

Nonimmune IgM

Reference DNA polymerasesPartially purified calf thymus a-polymerase (20 milliunits)t

Purified calf thymus p-polymerase (2 milliunits)Purified E. coli DNA polymerase I large fragment (30 milliunits)

190 ± 9115 ± 470 ± 440 ± 2

190 ± 9180-120115 ± 4110-7570 ± 465-4340 ± 2

40 ± 268 ± 4

DNApolymerase activity

recovered, milliunits*

Exp. 1 Exp. 2

0.070.30 0.370.020.02 0.070.040.03 0.020.020.60 0.15

Total 1.10 0.610000

0.380.320.190.300.230.260.22

Total 1.900.062.20

0.6

1.020.211.42

0.13.35

*Activity was measured with activated DNA as template-primer (19); 5-25 ,ul of each renatured enzyme solution wasincubated for 30 min in reaction mixtures with 50 ,uM [3H]dTTP (80,000 dpm/pmol). Activity calculated for the entiresolution is shown; 1 milliunit of activity = 1 nmol of dNMP (total) incorporated per hr at 37°C.

tImmunoprecipitates with MC pol 1; each extract was from 0.1 g of cells (50 milliunits of a-polymerase activity).SCommercial preparation of enzyme.

Biochemistry: Karawya et aL

7780 Biochemistry: Karawya et al.

Table 2. Specificity of electroeluted and renatured M, 190,000 DNA polymerase

Mr 190,000 Reference enzyme, %Modification polypeptide, % a-polymerase 3-polymerase

None 100 100 100With ddTTP (100 AM) 92 90 8

(500MAM) 58 64 0With aphidicolin (50 Ag/ml) 8 5 88With BuPhdGTP (1 MM) 5 10 80Without activated DNA; with (dT)" (rA)IO 34 140 2

Incubations were for 30 min with 20 Al of renatured enzyme solution. Native reference enzymeswere diluted in the renaturation dialysis buffer as described. Reaction mixtures (19) containedactivated DNA, 6 mM Mg2+, [3H]dTTP (80,000 dpm/pmol), and dNTP at 50MM. Incorporation valueswere proportional to the time of incubation and correspond (100%) to 1, 10, and 5 pmol of dNMP,respectively, for the Mr 190,000 polypeptide, a-polymerase, and f3-polymerase. BuPhdGTP, butyl-phenyldeoxyguanosine 5'-triphosphate; ddTTP, dideoxy TTP.

be noted that the renatured Mr 190,000 polypeptide had rela-tively less activity with poly(dT)-oligo(rA) than did the na-tive a-polymerase used as reference enzyme.

Activity Gel Analysis of Immunoprecipitated Polypeptides.Crude extracts from calf EBT cells or BSC-1 cells were sub-jected to immunoprecipitation as usual. Activity gel analysisthen was conducted with the immunoprecipitate and super-natant fractions (Fig. 2). Experiments with a second mono-clonal antibody, MC pol 2, were included for comparison.With both cell types, no signal from the Mr 190,000 polypep-tide was detected in the immunoprecipitate or correspondingsupernatant fraction. The MC pol 1 immunoprecipitate pro-duced strong signals at Mrs of 40,000, -70,000, and-115,000. Similar results were obtained with MC pol 2, ex-cept that the signal at Mr 40,000 was not observed in theimmunoprecipitate. The MC pol 1 immunoprecipitate andsupernatant fractions produced about equal signals in the Mr40,000 region; in experiments not shown, we found that high-er levels of MC pol 1 and second antibody failed to precipi-tate more of the Mr 40,000 activity. Immunoprecipitates pre-

1 2 3 4 5 6 7 8 Mr x 1O-3_._ *-Top

* 4p -15*

_0 _ _ _ -70

pared with nonimmune IgM failed to produce significant sig-nals in this experiment (not shown).The results indicate that the Mr 190,000 catalytic polypep-

tide in the immunoprecipitate could not be detected by activ-ity gel assay, whereas the Mrs 40,000, 70,000 and 115,000catalytic polypeptides produced clear signals. The failure toproduce an activity gel signal was true also for the catalyticpolypeptide of Mr 190,000 in the preparation of partiallypurified a-polymerase used in the electroelution and renatur-ation experiment in Table 1. This enzyme preparation pro-duced activity gel signals roughly corresponding to the re-sults in Table 1, except for the gel region between Mrs125,000 and 300,000 (Fig. 3).

DISCUSSIONIn this paper, we report the use of a monoclonal antibodyagainst calf a-polymerase to identify DNA polymerase targetpolypeptides in a crude extract of a growth-phase mammali-an cell line (BSC-1). The antibody immunoprecipitated DNApolymerase catalytic polypeptides that had been detectedpreviously in the crude extract both by activity gel analysisand by routine purification-i.e., Mrs of 40,000, 70,000, and115,000 (for discussion, see ref. 2). We found, however, thatpolypeptides corresponding to these three molecular weightswere relatively minor constituents of immunoprecipitatesprepared with extract from [35S]methionine-labeled cells.

Top-

Mrx 10-3

115-

W.70-

Pell Sn Pell SnEBT BSC-1

MCPol 1

Pell Sn Pell SnEBT BSC-1

MCPol2

FIG. 2. Autoradiogram showing results of activity gel analysis ofimmunoprecipitate (Pell) and supernatant (Sn) fractions preparedwith MC pol 1 or MC pol 2 and either calf EBT or monkey BSC-1cells. The immunoprecipitate from 60 Ml of extract, as indicated,was dissolved in NaDodSO4 sample buffer and mixed with a hetero-geneous protein mixture (24) prior to analysis in a NaDodSO4/10%polyacrylamide slab gel as described; proteins in the supernatantfraction were collected by acetone precipitation and then analyzedin the same way. Exposure for detection of 32P-labeled DNA prod-ucts was for 12 hr.

Dye .:Front 4

FIG. 3. Autoradiogram showing results of activity gel analysis ofthe partially purified calf thymus a-polymerase used in the experi-ment with reference DNA polymerases in Table 1. One milliunit ofenzyme was electrophoresed as in Fig. 2. Exposure was for 20 hr.

Proc. NatL Acad Sd USA 81 (1984)

40 *

40 Ablot.''

Proc. NatL Acad ScL USA 81 (1984) 7781

The major immunoprecipitated 35S-labeled component was ahigher molecular weight polypeptide of Mr 190,000 (Fig.1). This polypeptide was not active in our activity gel analy-sis of immunoprecipitates (Fig. 2); however, the polypeptideexhibited a-polymerase-like catalytic properties after elec-troelution from NaDodSO4/polyacrylamide gel and renatur-ation (Table 1). Hence, this Mr 190,000 catalytic polypep-tide, though quantitatively abundant, was unable to effi-ciently produce a signal in the activity gel assay, thusexplaining why activity gel analysis of crude extracts gener-ally has failed to reveal a strong signal in the Mr 190,000range (1-3, 13).

It is noted that a Mr 182,000 polypeptide was a main con-stituent of a-polymerase purified from Drosophila embryo(4-6), and the Korn group observed that a polypeptide ofMr

185,000 was a main constituent of their immunoaffinity-purified a-polymerase from KB cells (9). These higher mo-lecular weight polypeptides are probably analogous to thecatalytic polypeptide of Mr 190,000 described here. Thepresent results are the first demonstration that the mammali-an polypeptide of Mr 190,000 is indeed a DNA polymer-ase, and, by virtue of its catalytic properties, it is unequivo-cally identified as an a-polymerase. In light of these find-ings, questions about several aspects of a-polymerasestructure and function can be raised. For example, are thedifferent-size catalytic polypeptides in the crude extractprocessed from the Mr 190,000 polypeptide? It appears thatfurther work with MC pol 1 and other a-polymerase antibod-ies may answer this question. On the other hand, studies ofwhether the different-size catalytic polypeptides are in-volved in functionally different forms of holoenzyme proba-bly await development of suitable in vitro replication sys-tems.

Finally, a somewhat unexpected finding of the presentwork was that the a-polymerase monoclonal antibody MCpol I immunoprecipitated a Mr 40,000 catalytic polypeptide.Based upon its size, we assume that this polypeptide may bea l-polymerase; however, further studies are required to de-termine its precise identity. It should be noted that the possi-bility of obtaining antibody cross-reacting with a- and ,B-polymerases is well known (26, 27).

Note Added in Proof. Masaki et al. reported (28) use of their mono-clonal antibody to calf thymus a-polymerase for immunoprecip-itation of 10S a-polymerase from a crude extract of calf thymus.They observed an a-polymerase polypeptide of Mr 180,000 andspeculated that the higher molecular weight subunit of the 10S a-polymerase is synthesized as a protein of Mr 180,000.

W.A. was supported in part by a grant from the Deutsche For-schungsgemeinschaft (SFB 105).

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