Purification and properties of murine mammary tumor virus DNA polymerase

VIROLOGY

71, 242-254

Purification

(1976)

and Properties

STUART Memorial

L. MARCUS,

NURUL

Sloan-Kettering

Cancer

of Murine Mammary Polymerase H. SARKAR, Center,

Accepted

1275

January

York

AND Avenue,

Tumor

MUKUND New

York,

Virus DNA

J. MODAK New

York

7, 1976

Murine mammary tumor virus (MuMTV) DNA polymerase was purified by affinity chromatography on polycytidylate-agarose followed by phosphocellulose ion-exchange chromatography. The resulting enzyme preparation contained two major polypeptides, of 85,000 and 50,000 daltons as determined by SDS-polyacrylamide gel electrophoresis, and appeared, except for low levels of RNase H, free of DNase and RNase activity. The molecular weight of the purified native enzyme as determined by velocity sedimentation, was approx. 108,000. MuMTV DNA polymerase appears to be a zinc metalloenzyme and requires at least one reduced sulfhydryl group for the expression of catalytic activity. Apparent K, values determined for synthetic template-primers and deoxynucleoside triphosphates were 1 wg/ml and lo-12 PM, respectively. Examination of the optimal biochemical conditions for DNA synthesis on a variety of template-primers revealed that Mg’+ was the preferred divalent cation for DNA synthesis. Mn*+ could partially substitute for Mg’+, although it inhibited DNA synthesis when added in the presence of Mg’+. MuMTV DNA polymerase exhibited a preference for (dC),.(dG),,-,, among all the synthetic template-primers tested. Activated DNA was preferred as a heteropolymeric template for DNA synthesis when compared with viral 70 S RNA with either endogenous primers or with (dT),, as a primer. Rates of heat inactivation of the MuMTV DNA polymerase were found to vary depending upon the template-primer used to measure that inactivation. INTRODUCTION

ases, the amount of information on the properties of the B-type murine mammary tumor virus (MuMTV) enzyme is minimal (Howk et al., 1973; Dion et al., 1974 a, b). Apparent difficulties which were encountered in previous investigations on the nature of the B-type viral DNA polymerase were partly due to the lack of an adequate tissue-culture source of MuMTV. Colonies of high-tumor-incidence mice must be maintained in order for the virus to be purified from mouse milk (Dion et al., 1974a, b). Furthermore, MuMTV is found to be more resistant to disruption than Ctype viruses (unpublished observations), and the MuMTV DNA polymerase has been reported to be highly labile during purification (Dion et al., 1974b). We have previously reported the use of polycytidylate linked to agarose as an effective affinity matrix for the purification

The successful purification and biochemical characterization of oncornaviral RNA-directed DNA polymerase (reverse transcriptase) was first achieved using avian C-type oncornaviruses (Green and Gerard, 19741, due to the availability of large quantities of virus and the relative stability of their polymerases. Recently, the DNA polymerases of mammalian Ctype oncornaviruses have also been purified to near-homogeneity using modifications of the earlier procedures (Moelling, 1974; Gerard and Grandgenett, 1975; Verma, 1975). The structural and biochemical properties of the enzymes from avian and mammalian C-type oncornaviruses have also been extensively studied (Green and Gerard, 1974). In contrast to the mass of data available on the properties of C-type oncornaviral DNA polymer242 Copyright All rights

8 1976 by Academic Press, Inc. of reproduction in any form reserved.

MuMTV

DNA

POLYMERASE

243

poly(2’-O-methylcytidylate) was the kind gift of Dr. F. Rottman and was annealed to (dG),,,, as described above. Salmon sperm DNA was “activated” by limited exposure to DNase as previously described (Cavalieri et al., 1974). AMV 70 S RNA was prepared from purified virions as previously described (Modak et al., 1974). When AMV 70 S RNA was to be primed with (dT)l,,, annealing was performed just prior to use at an approximate molar ratio of 1:4. DNA polymerase assays. Reactions were carried out in a total volume of 0.1 ml and consisted of 50 m&f Tris-HCl (pH 7.8), 1 n-&f dithiothreitol (DTl!), and 20 pg of bovine serum albumin (fraction V). The deMATERIALS AND METHODS sired template-primer (synthetic) was added to a final concentration of 5 pglml. Viruses. Murine mammary tumor virus Divalent cation concentrations required (MuMTV), was purified from RI11 mouse milk as previously described (Sarkar and for optimal synthesis as MgCI, or MnCl, varied with the template-primer and are Dion, 1975). Avian myeloblastosis virus (AMV) was provided by Dr. J. Beard as indicated in the legends to figures and tables. Tritiated deoxynucleoside triphosplasma from infected chickens and purified as reported earlier (Marcus et al., phates were added together with unla1974a). Rauscher leukemia virus was pro- beled substrate to yield a final concentration of 20 PM with a specific activity of 250 vided through the courtesy of the Special cpm/pmole. Reactions were initiated by Virus Cancer Program by Dr. J. Gruber. Enzymes. Escherichia coli DNA polymthe addition of reaction mixture to enzyme erase I, as a homogeneous preparation, fractions and, unless otherwise indicated, was the kind gift of Dr. L. A. Loeb. were incubated for 30 min at 37”. Assays Rauscher leukemia-virus DNA polymerusing natural template-primers, e.g., actiase was purified from virions by affinity vated DNA and AMV 70 S RNA, contained chromatography using poly(rC)-agarose. 1 pg of RNA or 2.5 pg of DNA in place of Labeled compounds. Tritiated deoxynuthe synthetic template-primer. The reaccleoside triphosphates were purchased tion mixtures, in such cases, contained all from New England Nuclear, Inc. lz51 was four deoxynucleoside triphosphates, with purchased from Amersham-Searle, Inc. the three unlabeled precursors present at Template-primers. The template-primer 80 fl concentration and the labeled trinucleic acids used in the DNA polymerase phosphate at 10 PM concentration with a assays were purchased from P-L Biochemisp act of 1200 cpm/pmole. Reactions were cal Laboratories. Polyadenylic acid (rA), terminated by the addition of 5% (w/v) and polydeoxyriboadenylic acid (dA), were trichloroacetic acid solution containing annealed to oligodeoxythymidylic acid 0.01 M sodium pyrophosphate. Acid-insol(dT),,, before use at an equimolar nucleo- uble material was collected by vacuum filtide ratio by heating a solution of nucleo- tration onto Whatman glass fiber filters tides in 0.05 M Tris-HCI, pH 7.8, to 55” for (GF/B). After drying, the filters were 15 min and then allowing the solution to placed into toluene-based scintillation Cool slowly to room temperature. Polycytifluid and counted in a Packard liquid scindylic acid (rC), and polydeoxycytidylic acid tillation counter. (dC), were annealed to oligodeoxyguanylNuclease assays. RNase and DNase acate at equimolar ratios by heating the so- tivities were assayed by monitoring the lutions to 80” for 15 min, after which the degradation of tritiated polynucleotides to solutions were allowed to cool slowly to acid-soluble material. The substrate used room temperature. The synthetic template for detection of RNase H activity was of DNA polymerase from an avian C-type oncornavirus (Marcus et al., 1974a) and have successfully applied this technique to the purification of Rauscher leukemia-virus DNA polymerase (unpublished data). In this report we describe the purification of MuMTV DNA polymerase through the use of a combination of affinity and ionexchange chromatographic methods. The purified MuMTV enzyme was characterized with respect to molecular weight and apparent subunit composition, and the optimal conditions for DNA synthesis on a variety of natural and synthetic templateprimers were determined.

244

MARCUS,

SARKAR,

AND

MODAK

+X174 DNA-[3H]-RNA hybrid synthewas assayed using (dA),(dT),, as previsized as described by Verma (1975) with ously described (Cavalieri et al., 1974) and the following modifications. The reaction RLV DNA polymerase which was assayed mixture contained, in a final vol of 1.0 ml, with the same template-primers as the 80 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 MuMTV enzyme gradient. mM DTI’, 10 n&f MgC&, 100 pg of $X174 Labeling of viral proteins with Ir5Z. SoluDNA (Miles Laboratories), 1 mM each bilized virus or isolated polypeptides were ATP, CTP, and UTP, 20 PM of [3H]GTP iodinated using the lactoperoxidase proce(3000 cpm/pmole), and approx 100 pg of dure. To a l-ml solution containing viral homogeneous E. coli RNA polymerase. proteins 200-300 &i of [lz51]Na in 0.1 After 1.5 hr of incubation at 37”, the reacNaOH (Amersham) was added at room tion mixture was placed in a 70” water temperature. Subsequent steps were idenbath for 15 min, and quickly cooled in an tical to those described by Witte et al. ice bath. RNA-DNA hybrids were purified (1973). by isopycnic banding in cesium sulfate, Polyacrylamide-gel electrophoresis. Soand desalted by chromatography on a dium docecyl sulphate (SDS)-polyacrylSephadex G-50 column (Verma, 1975). The amide gel electrophoresis was performed reaction mixture for assay of RNase H ac- as described previously (Sarkar and Dion, tivity contained, in a vol of 0.1 ml, 20 mM 1975) with the following modifications. Tris-HCl (pH 7.8), 10 n-&? DTT, 150 mM Iodinated proteins were dialysed for 4-5 KCl, 0.02% (w/v) bovine serum albumin, days and then precipitated with 5% tri12 pmole of 4X174 DNA-13H]-RNA hybrid chloroacetic acid using 100 pg of bovine (as total nucleotides), and varying concenserum albumin as a carrier. The protein trations of Mg2+ or MI?+. To measure sin- precipitate was pelleted by centrifugation gle-stranded RNA degradation or DNase at 3000 g, and washed twice. The pellet activity 13Hlpoly(U) or (C), and was dissolved in 50 ~1 of 0.01 M sodium 13Hlpoly(dT), respectively, were used; re- phosphate buffer (pH 7.8), containing 1% action mixtures were identical to that de- (w/v) SDS and 1% (v/v) 2-mercaptoethanol. scribed for RNase H except that all con- After heating the samples at 70-80” for 15 tained 5 mM Mg2+ and 30 mM KCl. Reacmin, the proteins were electrophoresed in tions were initiated by the addition of en- gels containing 7.5% acrylamide for 16 hr zyme fraction and were incubated for varat 25 V, constant voltage. After running, ious times at 37”. Reactions were termigels were sliced into 2-mm fractions using a Gilson gel fractionator and lz51 radioacnated by the addition of trichloroacetic tivity determined using a gamma counter. acid, and acid-insoluble counts determined Preparation of poly(rC)-agarose. Polyas described in the DNA polymerase ascytidylate was covalently linked to agasay. Molecular weight estimations: Glycerol rose as previously described (Marcus et al., gradient. Velocity sedimentation was per1974a) and the resulting poly(rC)-agarose formed in preformed lo-30% (v/v) glycerol matrix contained approx. 0.3 mg poly(rC) per ml of packed agarose. All unreacted gradients in 0.01 M Tris-HCl buffer (pH groups left after CNBr activation were 7.8) containing 1 mM DTT and 0.4 M KCl. MuMTV DNA polymerase from peak col- blocked by washing the matrix with 0.1 M umn fractions was diluted fivefold in the glycine after the coupling had been performed. Failure to block unreacted groups same buffer used to prepare the gradients resulted in covalent linkage of some enand was layered over a 5-ml preformed gradient, which was then centrifuged for zyme protein together with other viral pro17 hr at 45,000 rpm at 4” in the SW 50.1 teins to activated, unsubstituted sites on rotor. Fractions were collected from the the agarose. Such nonspecific linkage may bottom of the tube and assayed for DNA result in considerable losses of enzyme acmatepolymerase activity with (rC)n-(dG)lZ-lB as tivity if small amounts of starting rial are used. well as (dC),*(dG),,-,e. Parallel gradients Disruption of MuMTV. The purified for molecular weight markers were run using E. coli DNA polymerase I, which MuMTV obtained through isopycnic band-

MuMTV

DNA

ing in sucrose gradients was pelleted and stored at -70” until use. The pellets were thawed and diluted with 0.05 it4 Tris-HCl (pH 7.8), to a protein concentration of approx. 6 mg/ml. To the virus suspension was added an equal volume of 2 x concentrated disruption buffer to yield a solution with the following components: 1% (v/v), Nonidet P-40 (NP-40), 0.5% (w/v), sodium deoxycholate, 0.05 M Tris-HCl (pH 7.8), 0.4 M KCl, and 10 mM dithiothreitol (DTT). The mixture was then allowed to stand in an ice bath for lo-20 min and was then centrifuged at 10,000 g for 20 min at 4”. Approximately 90-95% of available DNA polymerase activity measured using synthetic template-primers was found in the supernatant, which was diluted lo-fold with buffer containing 0.05 M Tris-HCl (pH 7.8), 1 m&f DTT, and 10% glycerol. This buffer composition was used throughout the purification procedure. Purification

of

MuMTV

DNA

245

POLYMERASE

thermore, it was found that incubation of partially disrupted virions at 37” for 30 min, as previously recommended (Dion et al., 1974b), inactivated the majority of enzyme activity. Greater than 90% of DNA polymerase activity was solubilized by the present procedure. The crude solubilized viral extract was applied to a poly(rC)agarose column which adsorbed 80-90% of input enzyme activity. The bound enzyme was eluted in a single step by the addition of 0.4 M KC1 in buffer. A typical elution profile of MuMTV DNA polymerase from a poly(rC)-agarose column is shown in Fig. 1A. The poly(rC)-agarose column fractions containing enzyme activity were then adsorbed onto a phosphocellulose column, and enzyme activity eluted with a linear gradient of increasing KC1 concentration as described in Materials and Methods. Elution carried outin this manner consistently resolved two peaks of DNA polymer-

polymer-

ase. All steps were carried out at 4” unless otherwise stated. Pasteur pipet columns (1.5 ml bed vol) of poly(rC)-agarose were poured prior to the beginning of puritication using glass wool as a bottom support and were equilibrated with buffer. The diluted, solubilized MuMTV supernatant was applied to the poly(rC)-agarose column and washed with buffer. Elution of adsorbed DNA polymerase activity was accomplished in one step using 0.4 M KC1 in buffer. The poly(rC)-agarose column eluate fractions containing MuMTV DNA polymerase activity were pooled, diluted lo-fold in buffer, and applied to a 15 x 0.9-cm phosphocellulose column which had been previously equilibrated with buffer. Bound enzyme was eluted with a 60 ml, O-O.6 M linear gradient of ascending KC1 concentration in buffer. RESULTS

Purification of MuMTV DNA polymerase. The conditions described in Materials

and Methods for MuMTV disruption and solubilization of enzyme activity yielded a crude starting material which possessed a specific activity lo- to 20-fold greater than that obtained by following the method previously published (Dion et al., 1974b). Fur-

180

I

/

1

I

I

/

FIG. 1. Purification of MuMTV DNA polymerase. (A) Chromatography of dilute, disrupted virus on poly(rC)-agarose. Fraction vol = 1.5 ml. Aliquots (5 ~11 of each fraction were assayed for DNA synthesis with (i-Cl.-(dG),,+,. Arrow indicates addition of 0.4 M KCl-containing buffer. Column flow rate = 16 ml/hr. (B) Chromatography of pooled poly(rCl-agarose column fractions containing DNA polymerase activity on phosphocellulose. Fraction vol = 1 ml. Aliquots (10 ~1) assayed for DNA polymerase activity as above. Flow rate = 18 ml/hr. Fractions designated under areas I and II were pooled and examined for polypeptide composition (Fig. 2).

246

MARCUS,

SARKAR.

ase activity (Fig. lB), although the relative areas under the peaks varied from one preparation to another. In some cases a single broad peak of enzyme activity was obtained. In order to determine the degree of purity of enzyme preparations obtained at each fractionation step, aliquots were removed and iodinated using the lactoperoxidase method (see Materials and Methods). The iodinated proteins were then examined by polyacrylamide-gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The polypeptide composition of solubilized MuMTV is shown in Fig. 2A. Five major proteins are resolved by this procedure, two of which, according to a previous report (Sarkar and Dion, 19751, are glycoproteins (gp55 and gp34) of molecular weights 55,000 and 34,000. The nonglycosylated proteins of molecular weights 28,000, 18,000, and 12,000 (~28, ~18, and ~121, have been designated constituents of the viral core. The initial step of poly(rC)-agarose column chromatography was found to remove the majority of viral proteins (Fig. 2B), and the major contaminant in this preparation was a 14,000 dalton polypeptide which can also be seen as a shoulder of the ~18 peak in the SDS-PAGE profile of the solubilized virus (Fig. 2A). It is interesting to note that such a protein has previously been observed to be a minor nonglycoprotein component of MuMTV (Sarkar and Dion, 1975). Both peaks from phosphocellulose appeared identical in their polypeptide composition (Fig. 2C, D) with the major protein appearing at a position corresponding to a molecular weight of 85,000, together with a 50,000-dalton protein, and minor components of 18,000 and 14,000 daltons. The enzymes from both phosphocellulose peaks possessedidentical properties in our hands, and may be considered as a single species. A summary of the purification procedure is given in Table 1. At least a go-fold increase in specific activity of the MuMTV DNA polymerase was achieved in the poly(rC)-agarose affinity chromatography step. The polypeptide composition of the pooled phosphocellulose column fractions suggests that significant purification was achieved by this step. Recover-

AND

MODAK

85

;h

,Dj 20

40

60

so

loo

120

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gels of iodinated proteins from (A) solubilized MuMTV; (B) pooled poly(rC)-agarose column eluate fractions; (C) phosphocellulose column DNA polymerase pool I (Fig. 1B); (D) phosphocellulose column DNA polymerase pool II (Fig. 1B). Proteins were iodinated and placed on 120-mm gels which were run, sliced, and counted (for ‘ziI-cpm) as described in Materials and Methods. The numbers above peak gel fractions correspond to molecular weight in thousands of daltons determined using marker gels with standard molecular weight proteins run at the same time as the experimental gels. Direction of protein migration is from left to right.

ies of input enzyme activity to 30%. Molecular

weight

varied from 20

of the active

enzyme.

The molecular weight of the native form of the purified MuMTV DNA polymerase was determined using velocity sedimentation through linear lo-30% glycerol gradients (see Materials and Methods). Rauscher murine leukemia virus (RLV) DNA polymerase and E. coli DNA polymerase I were used as molecular weight standards (Fig. 3). The active MuMTV enzyme was found to possessa sedimentation

MuMTV

DNA

value of 5.6 S, corresponding to a molecular weight of approx 108,000. MuMTV DNA polymerase activity always appeared to sediment as a single peak at all stages of purification. Nuclease activity. The purified MuMTV DNA polymerase preparations were found to be free of DNase and RNase (for singlestranded RNA) activity, measured using the appropriate 13Hlpolynucleotides as substrates (see Materials and Methods). RNase H activity was determined using 4x174 DNA-E3H]-RNA as a substrate under varying concentrations of Mg2+ and Mn2+. A recent study (Verma, 1975) has shown this substrate to be more effective

FIG. 3. Glycerol gradient centrifugation of puritied MuMTV DNA polymerase. Centrifugation was carried out at 45,000 rpm for 18 hr at 4”. MuMTV DNA polymerase (O-01 was located by assaying with (rC),.(dG),,_,,. An identical gradient contained 0.01 pg of homogeneous E. coli DNA polymerase I, which was located by using (dA),$dT),,, (A- -Al. RLV DNA polymerase was run as an additional marker and was located using (rC).$dG),,-,, (O-O). TABLE PURIFICATION Protein

source

OF MuMTV Volume

(ml) 1. Soluble, disrupted virions 2. Poly(rC)-Agarose column M KC1 peak fractions 3. Phosphocellulose pooled tions

pooled peak

0.4 frac-

40 10 15

247

POLYMERASE

in detecting RNase H activity associated with C-type viral DNA polymerases than homopolymeric RNA-DNA hybrid molecules. A low but detectable quantity of RNase H activity was found in purified MuMTV DNA polymerase preparations, equivalent to O.l-0.5% of the DNA synthetic activity observed using (rA),*(dT),, as template-primer. Preliminary studies indicate that the RNase H activity functions best in the presence of Mg*+. While this characteristic is similar to the preference for divalent cations exhibited by the H-II low-molecular-weight RNase H found in Moloney murine sarcoma-leukemia virus DNA polymerase preparations (Gerard and Grandgenett, 19751, our recovery of enzyme activity from sedimentation velocity experiments has been too low to allow a determination of the molecular weight of the RNase H activity we have observed. Quantities of enzyme available were not sufficient to allow complete characterization of the RNase H activity, and therefore the question of whether it is an intrinsic activity of MuMTV DNA polymerase or a cellular contaminant present at low levels which copurified with the enzyme requires further study. Requirements for DNA synthesis. Little has been reported regarding the ability of MuMTV DNA polymerase to use a variety of natural and synthetic template-primers for DNA synthesis. Conditions reported as optimal for DNA synthesis have in general been determined using either detergentdisrupted virions (Dion et al., 1974a; Wu and Cetta, 1975) or preparations of MuMTV DNA polymerase which possessed nuclease activity and considerable 1 DNA

POLYMERASE

Total protein (nip) 12.3 co.2 ND

Total

activit@

Specific tivitp

ac4.6

Yield (%)

57,000 49,000

>245

100 86

12,500

ND

22

n Total Activity is expressed as Total Units. One unit is defined as the amount of enzyme catalyzing the incorporation of 1 pmole of 13HldGMP into acid-insoluble material in (rC),.(dG),,-,,-directed DNA synthesis in 30 min at 37”. Specific activity is expressed as units per microgram of protein. ND, Not determined.

248

MARCUS,

SARKAR,

heterogeneity (Dion et aZ., 1974131,with respect to protein composition. In this study, we have examined the various biochemical parameters affecting DNA synthesis by partially purified preparations of MuMTV DNA polymerase and have determined the individually optimal conditions for such synthesis on a number of templateprimers. The results of these studies are described below. Divalent cation requirements. An absolute requirement for divalent cations was exhibited by the MuMTV DNA polymerase on all template-primers tested. Except for (rCm),*(dG)12-18, Mg’+ was the preferred divalent cation for all of the templateprimers examined (Table 21, and Mn*+ could only partially substitute for Mg2+, producing up to 25% of the optimum rates of synthesis. The concentrations of Mn2+ and Mg2+ listed in Table 2 were those UTILIZATION

OF VARIOUS

Template-primer Synthetic (rA)..(dT),,, (rA1, (dA)..(dT),,, (d’h, (rC)..(dG),,-,, (rCml.*(dG),,-,, (dC)..(dGl,,-,, Natural Activated AMV AMV

salmon 70 S RNA* 70 S RNA

sperm

+ (dT),,,

DNA

AND

MODAK

found to be optimal for DNA synthesis on each respective template-primer. The degree to which Mn2+ could substitute for Mg2+ also varied with the template-primer used to direct DNA synthesis (Table 2). With Mg2+ as divalent cation, the concentrations required for optimal rates of DNA synthesis were found to differ depending upon the template-primer directing the synthesis (Fig. 4). Considerable variation in the optimal Mg2+ concentrations was observed, as in (rA);(dT),,-directed synthesis (2 m&f Mg2+) compared with (r-Cl,; (dG)12-,8-directed DNA synthesis (30 mM Mg2+). Much less variation was observed for those concentrations of Mn2+ which supported optimal rates of DNA synthesis on the various template-primers. Optimal concentraions of Mn2+ varied from 0.05 to 0.2 mA4. Template-primer

TABLE TEMPLATE-PRIMERS

utilization.

The rela-

2 BY MuMTV

DNA

POLYMERABE”

Divalent

cation

Wtf)

pmole Incorporation

Mg’+ MnZ+ MgZ+ Mg’+ Mg’+ Mg’+ Mn2+ MgZ+ Mn’+ Mgl+ Mn’+

(2) (0.05) or Mn2+ or Mn’+ or Mn’+ (30) (0.1) (30) (0.1) (10) (0.2)

TTP TTP TTP TTP TTP dGTP dGTP dGTP dGTP dGTP dGTP

Mg’+ Mnr+ Mg”+ Mg*+ Mg%+ Mg2+

(30) (0 2) (5; (5) (51 - dCTP (51 - dATP

dGTP dGTP dGTP dGTP dGTP dGTP

220 60
a Assays were performed as described in Materials and Methods. Divalent cations were added individually as desired. All incubations were at 37” for 30 min. Identical amounts of enzyme were used in all reactions. For those reactions in which synthesis was directed by a naturally occurring template-primer, the picomoles incorporated are given only for the labeled substrate although all four deoxynucleoside triphosphates were present. A constant 16-20 cpm background level of incorporation was subtracted from all results. b The same preparation of 70 S RNA was also tested for its ability to direct DNA synthesis catalyzed by AMV DNA polymerase. Under conditions identical to those used for the MuMTV enzyme, a quantity of AMV DNA polymerase allowing the incorporation of 1500 pmoles of [3HldTMP in (rA),.(dTl,,directed synthesis also catalyzed, in the presence of all four DNA precursors, the 70 S RNA-directed incorporation of 4 pmole of 13HldGMP.

MuMTV

VI, Oo 5

IO

I 20 Mg”

I 30 (mbl)

I 40

DNA

. \-I hRNA J 50

FIG. 4. Determination of Mg2+ optima for MuMTV DNA polymerase activity using various template-primers. Assays were performed as described in Materials and Methods except for varying the concentration of Mg*+. Values corresponding to 100% activity in this experiment (as picomoles appropriate precursor incorporated) were: (rC),. (dG),,-,, = 120; (dC).$dG),l-,, = 325; (rA),. (dT),, = 100; activated DNA = 10; AMV 70 S RNA + (dT),, = 0.4.

tive ability of MuMTV DNA polymerase to carry out DNA synthesis on a variety of natural and synthetic template-primers at optimal Mg2+ or MnZ+ concentrations is shown in Table 2. Incorporation of DNA precursors complementary to polymeric templates required the presence of an initiator or “primer” molecule annealed to the template, exemplified by the data given in Table 2 with (r-A), and Noncomplementary nucleo(rA);WI’h,,. tides were not incorporated (data not shown) to any significant degree. Among the synthetic homopolymeric templateprimers examined, (dC),*(dG)lz-le was the most efficiently copied, yielding rates of DNA synthesis three-fold greater than those observed with (rC);(dG),,-,,. Commercially available batches of (rC),(dG),,-,, were found to vary considerably in their ability to direct DNA synthesis. However, freshly annealed templateprimers prepared just prior to use were quite satisfactory and yielded reproducible results. Although (rA),*(dT),, was effective as a template-primer, (dA),*(dT),,, did not appear to direct DNA synthesis with either Mg2+ or MnZ+ as divalent cation. The synthetic template-primer poly(2’-Omethylcytidylate).oligodeoxyguanylate [(rCm),*(dG),,-,,I has been reported to be

POLYMERASE

249

an effective template-primer for many reverse transcriptases (Green and Gerard, 1974), and to be highly specific for detecting the presence of oncornaviruslike DNA polymerases in human tissues (Gerard, 1975). In agreement with these studies, the purified MuMTV DNA polymerase was found to utilize this template-primer although the resulting rates of DNA synthesis were &fold lower than those obtained with (rC),*(dG),,-,, as template primer. Of the two natural template-primers, Salmon sperm DNA “activated” by limited treatment with DNase I (Cavalieri et al., 1974) was greately preferred by the MuMTV DNA polymerase over AMV 70 S RNA (Table 2). Addition of a (dTl,,, primer enhanced DNA synthesis on AMV 70 S RNA. That heteropolymeric rather than homopolymeric regions of RNA were copied by the MuMTV enzyme is shown by the requirement for all four DNA precursors (Table 2). K, for template-primers and substrates. Apparent K, values were determined by the double-reciprocal plot method for various synthetic template-primers and their appropriate deoxynucleoside triphosphates. Values of 1 pg/ml +- 20% and lo-12 $l4 for template-primers and DNA precursors, respectively, were obtained. pH optima. Using (rA);(dT),,,, (rC); (dG)lP-lS, and (dC),*(dG),,-,, as templateprimers, the MuMTV DNA polymerase exhibited a pH optimum of 7.8 (Fig. 5). Similar results were obtained for natural template primers (data not shown). Temperature optima. Previous studies using C-type oncornaviral DNA polymerases (Waters and Yang, 1974) indicated that temperature optima for DNA synthesis were both template- and enzyme-specific. The data expressed in Fig. 6 shows the temperature response of MuMTV DNA polymerase with (rC);(dG),,-,,, kWt.(dGL,, and (rA),.(dT),,, as template-primers. For both (i-A),- and (dC),directed DNA synthesis an optimum incubation temperature of 37” was observed while (rC),-directed DNA synthesis functioned best at 42”. The patterns of the MuMTV DNA polymerase response to incubation temperature also appeared to vary with the template-primer, and may

250

MARCUS,

SARKAR,

FIG. 5. Determination of pH optima for MuMTV DNA polymerase activity directed by (dC)..(dGl,,-,,, (rC)..(dG),,-,,, and (rA),.(dT),, at individually optimal Mg*+ concentrations (given in Table 2). Tris buffer 0.05 M adjusted to the appropriate pH with HCl was used in all cases. The 100% values for picomoles of appropriate precursor incorporated are identical to those shown in Fig. 4.

AND

MODAK

poor substitute for Mg2+ in reaction mixtures, Mn2+ was found to inhibit DNA synthesis by the MuMTV enzyme when added to reaction mixtures containing Mg2+. The addition of monovalent cations to reaction mixtures containing Mg2+ also caused inhibition of DNA synthesis. Potassium phosphate buffer (pH 7.8) did not dramatically affect DNA synthesis on either template-primer, in marked contrast to results obtained with C-type oncornaviral DNA polymerases (unpublished data). The addition of N-ethyl maleimide to MuMTV DNA polymerase completely inhibited DNA synthesis on both templateprimers, indicating a requirement for reduced sulfhydryl groups at the active site of the enzyme. The inhibition of DNA synthesis as a result of 0-phenanthroline addition suggests that the MuMTV DNA polymerase, like other oncornaviral DNA polymerases (Green and Gerard, 1974), is a zinc-containing metalloenzyme. A previous report (Wu and Cetta, 1975) indicated that the nonionic detergent Triton X-100, when added to reaction mixTABLE

3

EFFECT OF EXOGENOUS ADDITION OF VARIOUS COMPOUNDS ON (rA),.(dT),, AND (rCl,~(dGl,,-,8DIRECTED DNA SYNTHESIS BY MuMTV DNA PoLYMERA~EO Addition 0

eo

30 lncubolmn

40 Ter%perotute

50

60

FIG. 6. Determination of temperature optima for MuMTV DNA polymerase activity by (dC1,. and (rA),(dTl,,. Assays (dG),,-,,, (rC).(dG),,-,,, were performed as described in Materials and Methods and incubated at the appropriate temperature for 20 min. Optimal Mg2+ concentrations were used for each template-primer (See Table 2). Values for 100% activity expressed as picomoles of appropriate incorporated: (rA).(dT),, = 100; precursor (rC)R.(dG),2-,8 = 170; (dC),~(dGl,,-,a = 325.

relate to the stability of the templateprimer duplex (Waters and Yang, 1974). Effect MuMTV

pmole 3H-substrate rated

incorpo-

(‘C)

of various compounds DNA polymerase activity.

on

The addition of various compounds to reaction mixtures and their effect on MuMTV DNA polymerase activity is shown in Table 3, using (rA);(dT) 10 and (rC),*(dG),z_,8 as template-primers. In addition to being a

(rA)..(dT),, None Mn2+ (0.2 n&f) KC1 or NaCl (50 n-&f) N-Ethyl maleimide (1 mM) 0-phenanthroline (1 mM) Potassium phosphate (pH 7.8) (10 mkfl Triton X-100 0.02% (wi v) Triton X-100 0.05% (wl v)

100 40 75 1 0.5

(rC):(dG),,,, 200 20 150 0.5 0.5

80

180

110

200

100

200

a Assays were performed as described in Materials and Methods, with the above compounds added to the indicated final concentrations as shown. Mg*+ was used at 2 mJcf for (rA),*(dT),,-directed reactions, and at 30 mM for (rC),.(dG),2-,,-directed reactions. Incubations were at 37” for 30 min.

MuMTV

DNA

tures containing synthetic templateprimers, caused several-fold stimulation of DNA synthesis catalyzed by a variety of viral DNA polymerases. We found (Table 3) that the addition of Triton X-100 to reaction mixtures containing purified MuMTV DNA polymerase had little or no effect on the amount of product synthesized. Enzyme stability and heat inactivation. Purified MuMTV DNA polymerase preparations, which were stored at -70” in the presence of 0.1% (w/v) bovine serum albumin and 1 mM DTT in 10% (v/v) glycerol containing 0.05 M Tris-HCl (pH 7.8), retained greater than 90% activity over a 3month period when tested with synthetic and natural template-primers. A previous study using partially purified MuMTV DNA polymerase (Dion et al., 1974b) indicated that differential labilities were exhibited upon storage, with (rC),*(dG),,,,directed DNA synthesizing ability more labile than activated DNA-directed synthetic ability. In an earlier study (Marcus et al., 1974b), using a variety of cellular and C-type oncornavirus DNA polymerases, we reported that the rate of heat inactivation of a DNA polymerase, heated in the absence of substrate or templateprimer, varied depending upon the template-primer used to measure that inactivation. A similar analysis of the kinetics of heat inactivation was performed using the MuMTV enzyme. The purified MuMTV DNA polymerase was incubated at 51” for various periods of time in the absence of reaction components and the rate of heat inactivation then measured using @A),+ (dTL1, (rC),+lG),,-,,, (dC),*(dGh-ls, and activated DNA as template-primers. The results of this experiment are shown in Fig. 7. Differences in rates of inactivation were observed with the different templateprimers, with (d&-directed DNA synthesis being the most labile. DISCUSSION

We have described the purification of MuMTV DNA polymerase, using poly(rC)-agarose and phosphocellulose chromatography. The low amounts of protein recovered necessitated the use of iodination techniques in order to estimate the polypeptide composition of the purified

POLYMERASE

251

10 30 50 50 r

+0 30 1 ?OC

FIG. 7. Heat inactivation of MuMTV DNA polymerase at 51” measured using various templateprimers. Purified MuMTV DNA polymerase in buffer containing 0.05 M Tris-HCl (pH 7.8), 1 mM DTT, 10% (v/v) glycerol, and 0.1% bovine serum albumin was placed in a 51” water bath for different periods of time, after which aliquots were removed and cooled in an ice bath. Ice-cold reaction mixtures containing various template-primers were added and DNA synthesis initiated by placing the assay tubes in a 37” water bath. Reactions were carried out for 20 min at 37”. The percent activity remaining for all reactions was calculated using as controls enzyme fractions which had not been exposed to heat.

DNA polymerase preparations (Fig. 2). The purified MuMTV DNA polymerase preparations contained two major polypeptides, of 85,000 and 50,000 daltons, and two minor (~10%) polypeptides, of 18,000 and 14,000 daltons (Fig. 2C, D). The molecular weight of the active forms of the enzyme, as determined by sedimentation velocity analysis, was 108,000 (Fig. 3). The differences observed in the molecular weights of the active forms of the enzyme and the largest molecular weight polypeptide present in our partially purified preparations suggests that the MuMTV enzyme may possess a subunit structure, although it is not possible at this time to determine whether the active enzyme is a homopolymer or heteropolymer. Studies

252

MARCUS,

SARKAR,

are in progress to clarify the true subunit composition of this enzyme. The MuMTV DNA polymerase preparations contained no detectable DNase or single-stranded RNA-degrading activity, although a low level of RNase H activity was found which preferred Mg2+ as divalent cation. It is not yet known whether this activity is an intrinsic function of the MuMTV DNA polymerase. The purified MuMTV DNA polymerase was found to be capable of carrying out DNA synthesis on a variety of natural and synthetic template-primers (Table 2), and requires a sulfhydryl reducing agent for activity. The most efficiently utilized template-primer was found to be (dC); (dG),,-,,. DNA synthesis directed by (rC), with the same primer molecule resulted in rates of synthesis which were only onethird those observed with (dC),*(dG)12-18 (Table 2). The synthetic template-primer which has been claimed to be “diagnostic” for the identification of oncornaviral DNA polymerase, Wm),WL18 Gerard, 1975), is very poorly utilized by MuMTV DNA polymerase when compared with the templates (dCX and (rC), (Table 2). The low level of (rCm), utilization by the MuMTV enzyme may still be significant in light of the fact that cellular R-DNA polymerase, which possess template-primer preferences similar to mammalian viral reverse transcriptase (Spadari and Weissbath, 1974; Lewis et&., 1974), cannot utilize (rCm), as a template (Gerard, 1975). Of the natural template-primers tested, activated DNA was much preferred as a template when compared with AMV 70 S RNA for complementary DNA synthesis. When (dT),, was annealed as a primer molecule to 70 S RNA and TTP used as labeled substrate in the presence of the other DNA precursors, a 20-fold stimulation of synthesis was observed (data not shown). This increase was disproportionate to that which had been observed using [3H]dGTP as the labeled substrate (Table 2) and further studies indicated that TTP incorporation occurred in the absence of other DNA precursors and was dependent on the presence of the (dT),, primer. Therefore, it appears that the high level of dTTP incorporation which we observe in the presence of

AND

MODAK

the (dT),, primer is due to synthesis of the homopolymer poly(dT). In the absence of (dT),, and in the presence of all four DNA precursors, dTTP incorporation is approximately equivalent to dGTP incorporation. Wu and Cetta (1975) reported that the nonionic detergent Triton X-100 stimulated DNA synthesis by several oncornaviral DNA polymerases directed by synthetic template-primers. In contrast to this report, we find (Table 3) using purified MuMTV DNA polymerase and individually optimal conditions for synthesis on each template-primer, that addition of Triton X-100 to reaction mixtures had no effect on the rate of DNA synthesis by this enzyme. Our results suggest that the previously reported stimulation of MuMTV DNA polymerase by detergent may have been due, in part, to the impure enzyme preparations used and the lack of optimal reaction conditions. It is also important to note that monovalent cations, which are added routinely to reaction mixtures of various oncornaviral DNA polymerases (Green and Gerard, 1974) including MuMTV (Dion et al., 1974a, b), inhibit DNA synthesis carried out at optimal divalent cation concentrations (Table 3) by the purified MuMTV enzyme. Dion et al. (1974b) reported that partially purified MuMTV DNA polymerase, upon storage, preferentially lost RNA-directed DNAsynthesizing ability. We have found that all DNA-synthesizing activities of the MuMTV enzyme remain stable when frozen for long periods of time (over 3 months) at -70” in the presence of appropriate protectants (bovine serum albumin, glycerol, and DTT). Heating MuMTV DNA polymerase in the absence of reaction mixture components produced different rates of inactivation depending upon the template-primer used to measure that inactivation (Fig. 7). This finding is consistent with the hypothesis that different template-primers may induce different conformative responses by DNA polymerases, or that template-specific subsites may exist on these enzymes, and similar results have been reported for a number of cellular and oncornaviral DNA polymerases (Marcus et al., 1974b). MuMTV DNA polymerase has previ-

MuMTV

DNA

ously been reported to be similar in its biochemical properties to the DNA polymerase purified from Mason-Pfizer monkey tumor virus (MPMV), which was derived from a primate mammary adenocarcinoma and which has properties of both B-type and C-type oncornaviruses (Kramarsky et al., 1971). While immunologically dissimilar (Yaniv et al., 19741, both the MuMTV and MPMV enzymes prefer Mg2+ as a divalent cation over Mn2+ and appear to have the same molecular weight in the active form (Abrell and Gallo, 1973; Dion et al., 1974a). The MPMV DNA polymerase, however, has been shown to be a single polypeptide (Abrell and Gallo, 1973), while our results indicate that the MuMTV DNA polymerase may possess a multiple-subunit structure. We are currently investigating the comparative biochemical and biophysical properties of various C-type oncornaviral DNA polymerases as well as the MPMV enzyme in order to determine functional and structural similarities and differences. The MuMTV DNA polymerase exhibits a deficiency in the ability to copy natural RNA templates as compared with activated DNA. Although similar results have been reported for some mammalian C-type viral DNA polymerases, preliminary studies indicate that the MuMTV enzyme is only l-10% as effective in copying natural RNA templates as MuLV DNA polymerases. We are currently examining the possible existence of viral or cellular factors which might serve to stimulate natural RNA copying by the MuMTV enzyme. Another question which remains to be answered is whether the low levels of RNase H activity which we find copurifying with the MuMTV DNA polymerase are an intrinsic property of the enzyme, as has been reported for murine leukemia virus DNA polymerase (Gerard and Grandgenett, 1975; Verma, 1975), or whether this activity represents contamination from cellular sources. ACKNOWLEDGMENTS The authors wish to thank Drs. P. J. Gomatos and L. J. Old for encouragement. The technical assistance of Steven W. Smith and Emerson S. Whittington is appreciated. This work was sup-

253

POLYMERASE ported, in part, CA-17129.

by NC1

Grant

Nos.

CA-08748

and

REFERENCES ABRELL, J. W., and GALLO, R. C. (19’73). Purification, characterization, and comparison of the DNA polymerases from two primate RNA tumor viruses. J. viroz. 12, 431-439. CAVALIERI, L. F., MODAK, M. J., and MARCUS, S. L. (1974). Evidence for allosterism in in vitro DNA synthesis on RNA templates. Proc. Nut. Acad. Sci. USA 71, 858-862. DION, A. S., VAIDYA, A. B., and FOUT, G. S. (1974a). Cation preferences for poly(r0oligo (dG)-directed DNA synthesis by RNA tumor viruses and human milk particulates. Cancer Res. 34, 3509-3515. DION, A. S., VAIDYA, A. B., FOUT, G. S., and MOORE, D. H. (1974b). Isolation and characterization of RNA-directed DNA polymerase from a Btype RNA tumor virus. J. Viral. 14, 40-45. GERARD, G. F. (1975). Poly(2’-O-methylcytidylate).oligodeoxyguanylate, a template primer specific for reverse transcriptase, is not utilized by HeLa cell R-DNA polymerases. Biochem. Biophys. Res. Commun. 63, 706-711. GERARD, G. F., and GRANDGENETT, D. P. (1975). Purification and characterization of DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. J. Viral. 15, 785-797. GREEN, M., and GERARD, G. F. (1974). RNA directed DNA polymerase -Properties and functions in oncogenic RNA viruses and cells. Prog. Nucleic Acid Res. Mol. Biol. 14, 187-334. HOWK, R. S.,RYE, L. A., KILLEEN, L. A., SCOLNICK, E. M., and PARKS, W. P. (1973). Characterization and separation of viral DNA polymerase activities in mouse milk. Proc. Nut. Acad. Sci. USA 70, 2117-2121. KRAMARSKY, B., SARKAR, N. H., and MOORE, D. H. (1971). Ultrastructural comparison of a virus from a rhesus monkey mammary carcinoma with four oncogenic viruses. Proc. Nut. Acud. Sci. USA 68, 1603-1607. LEWIS, B. J., ABRELL, J. W., SMITH, R. G., and GALLO, R. C. (1974). Human DNA polymerase III (R-DNA polymerase): Distinction from DNA polymerase I and reverse transcriptase. Science 183, 867-868. MARCUS, S., MODAK, M. J., and CAVALIERI, L. F. (1974a). Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose. J. ViroE. 14, 853-859. MARCUS, S. L., MODAK, M. J., and CAVALIERI, L. F. (1974b). Evidence for template-specific sites on DNA polymerases. Biochem. Bioihys. Res. Commm. 56, 516-621. MODAK, M. J., MARCUS, S. L., and CAVALIERI, L. F. (1974). Synthesis of DNA complementary to AMV 70 S RNA by E. coli DNA polymerase I. Biochem. Biophys. Res. Commun. 56, 247-255.

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MOELLING, K. (1974). Characterization of reverse transcriptase and RNAse H from Friend-murine leukemia virus. Virology 62, 46-59. PARKS, W. P., HOWK, R. S., SCOLNICK, E. M., OROSZLAN, S., and GILDEN, G. (1974). Immunochemical characterization of two major polypeptides from murine mammary tumor virus. J. Virol. 13, 12001210. SARKAR, N. H., and DION, A. S. (1975). Polypeptides of the mouse mammary tumor virus. I. Characterization of two group specific antigens. Virology 64, 471-491. SPADARI, S., and WEISSBACH, A. (1974). HeLa cell Rdeoxyribonucleic acid polymerases. Separation and characterization of two enzymatic activities. J. Biol. Chem. 249, 5809-5815. VERMA, I. M. (1975). Studies on reverse transcriptase of RNA tumor viruses. III. Properties of purified Moloney murine leukemia virus DNA polym-

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erase and associated RNAse H. J. Viral. 15, 843854. WATERS, L. C., and YANG, W. K. (1974). Comparative biochemical properties of RNA directed DNA polymerases from Rauscher leukemia virus and avian myeloblastosis virus. Cancer Res. 34, 25852593. WITTE, 0. N., WEISSMAN, I. L., and KAPLAN, H. S. (1973). Structural characteristics of some murine RNA tumor viruses studied by lactoperoxidase iodination. Proc. Nat. Acad. Sci. USA 70,36-40. WV, A. M., and CETTA, A. (1975). On the stimulation of viral DNA polymerase activity by nonionic detergent. Biochemistry 14, 789-795. YANIV, A., OHNO, T., KACIAN, D., COLCHER, D., WITKIN, S., SCHLOM, J., and SPIEGELMAN, S. (1974). Serological analysis of reverse transcriptase of the Mason-Pfizer monkey virus. Virology 59, 335-338.