THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc. Vol. 260, No. 10, Issue of May 25, pp. 6254-6263,1985

Printed in U.S.A.

DNA Primase-DNA Polymerase a! from Simian Cells MODULATION OF RNA PRIMER SYNTHESIS BY RIBONUCLEOSIDE TRIPHOSPHATES*

(Received for publication, October 18, 1984)

Masamitsu Yamaguchi, Eric A. Hendrickson, and Melvin L. DePamphilis From the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02215

DNA primase-DNA polymerase a, purified 53,000- fold from CV- 1 cells, synthesized predominantly (p)ppA(pA)s-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (P)PPA/G(PN)~-,, and 20% were (p)ppA/G(pN)~,_~. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3‘-5‘ exonuclease, leaving a single dNMP at the 3‘-end of the RNA primer, and the terminal 5’-(p)ppN residue was determined by “capping” with [ ~ U - ~ ~ P I G T P using vaccinia guanylyl- transferase. The processivity of DNA synthesis initi- ated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G , even at high levels of CTP or UTP, although the ratio of A to G varied from 4:l to 1:l depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides a t RNA covalently linked to the 5’-end of DNA (RNA-p-DNA) junctions, and in- creasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthe- sized by DNA primase-DNA polymerase a is modulated by the relative concentrations of ribonucleoside tri- phosphates.

DNA polymerase a appears to be solely responsible for DNA synthesis on both sides of replication forks in eukaryotic cells and papovavirus chromosomes (1, 2). However, this enzyme is associated specifically with other proteins that modify its activity. Stimulatory proteins C1C2 greatly enhance the ability of a-polymerase to initiate synthesis on extensively single-stranded DNA templates containing either RNA or DNA primers (3,4). DNA primase synthesizes an RNA primer on which a-polymerase can initiate synthesis on DNA tem- plates. DNA primase activity has been detected in extracts of CV-1 cells ( 5 ) , human lymphocytes (6), Xenopus eggs (7, 8) , and Drosophila embryos (9), and it co-purifies with DNA polymerase a from mouse cells (10, 12), human cells (13, 18), calf thymus (14), Drosophila embryos (15, 16), Xenopus eggs (17), and with DNA polymerase I (the homologue of a- polymerase) from yeast (19, 20). Furthermore, DNA primase

*This work was supported by grants from the National Cancer Institute and the American Cancer Society The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

activity in mouse (10) and human (13) cells can be immuno- precipitated with monoclonal antibodies against a-polymer- ase. When the primase-polymerase complex is provided with a DNA template and the complementary rNTP and dNTP substrates, it synthesizes RNA-primed DNA in a fashion characteristic of DNA synthesis in uiuo: synthesis is resistant to a-amanitin, and an oligoribonucleotide, 7-12 bases long, beginning with A or G is found covalently attached to the 5’- ends of nascent DNA chains. Thus, it appears that a DNA primase-DNA polymerase a complex is responsible for initi- ation of Okazaki fragments a t eukaryotic replication forks.

Our objective was to purify DNA primase-DNA polymerase a from CV-1 cells, the permissive host for simian virus 40 (SV40), and attempt to accurately reconstruct SV40 DNA replication in uitro. The events at SV40 and polyoma virus replication forks appear homologous to those in mammalian chromosomes (1,2,21). CV-1 cells were chosen in anticipation of cell-specific interactions between viral T-antigen and cel- lular proteins that may be involved in the synthesis of RNA primers at the origin of replication (22,27), as well as between different proteins within a replication complex (e.g. ClC2 and a-polymerase (4)). The size and 5’-terminal nucleotide com- position of RNA primers synthesized on different templates was determined, as well as the effects of changing the relative concentration of each rNTP’ in the reaction mixture. DNA primase-DNA polymerase a synthesized primers about 3 bases shorter than synthesized i n uiuo. However, the 5”terminal ribonucleotide selected by the purified primase-polymerase complex was determined by the relative concentrations of ATP and GTP, similar to the synthesis of polyoma virus RNA-primed DNA chains in isolated nuclei (58).

EXPERIMENTAL PROCEDURES~

RESULTS

Purification of D N A Primase-DNA Polymerase a-DNA primase-DNA polymerase (Y activity from CV-1 cells was purified on the basis of its ability to incorporate the comple- mentary dNTP into a poly(dC) or poly(dT) template only when the complementary rNTP was also present. The enzyme

The abbreviations used are: rNTP, ribonucleotide triphosphate; SDS, sodium dodecyl sulfate; dNTP, deoxyribonucleoside triphos- phate; T4 exo, phage T4 DNA polymerase 3’-5‘ exonuclease; PEI, polyethyleneimine; RNA-p-DNA, an RNA molecule covalently linked by a phosphodiester bond to the 5’-end of the molecule.

“Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3221, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

6254

DNA Primase-DNA Polymerase a from Simian Cells 6255

TABLE I Purification of DNA primae-DNA polymerase (Y

Fraction

DNA synthesis (units)

Protein +rNTPs -rNTPs F'urification, poly(dC)

poly(dC) PoMdT) Activated DNA mg" % units/mg

I (extract) 1210 4,640 (100) 1,650 (100) 29,700 (100) 4 1 I1 (DEAE-cellulose) 1070 4,450 (96) 1,670 (100) 29,700 (100) 4 1 I11 (AmSO,; DEAE- 77 3,750 (81) 951 (58) 26,100 (88) 49 13

IV (Sepharose CL-GB) 33 2,940 (63) 473 (29) 15,700 (53) 89 23 V (hydroxylapatite) 5 2,640 (57) 339 (21) 11,000 (37) 530 140 VI (DEAE-Bio-Gel)

A 3.5 3,910 (84) 363 (22) 6,420 (22) 1,100 290 B 1.4 154 (3) 22 (1) 3,310 (11) 110 29

A 0.16 1,130 (24) 126 (8) 2,080 (7) 7,000 1,800 '

B 0.24 88 (2) 13 (1) 1,540 (5) 360 95

Sephacel)

VI1 (butyl-agarose)

VI11 (DNA-Sepharose; poly(r1)-agarose)

A 0.020 1,180 (25) 127 (8) 1,514 (5) 59,000 16,000 A (peak fraction) 0.0026 515 (11) 636 (2) 200,000 53,000 B 0.032 182 (4) 18 (1) 938 (3) 5,700 1,500

"Quantities are per 100 g, wet weight, of CV-1 cells. Protein was measured by UV absorption, except for Fractions VI11 which were determined by densitometry of stained gels (e.g. Fig. 2). A standard curve was constructed by similar gel electrophoresis of known quantities of several protein standards.

TABLE I1 Enzymatic activities in purified DNA primase-DNA polymerase (Y

Fraction VIII-A (Table I). Enzyme" Activity

DNA primase (poly(dC)) 1.6 X 10' pmol dNMP/h DNA polymerase (activated DNA) 2.1 X lo3 pmol dNMP/h ATPase

-ssDNAb <0.04 pmol ADP/h +ssDNA C0.04 pmol ADP/h

5"3' 0.04 pmol dNMP/h 3 ' 4 ' 0.07 pmol dNMP/h

Exo- and endonuclease (ssDNA) 0.4 pmol dNMP/h Endonuclease (dsDNA) CO.01 pmol dNMP/h e See "Experimental Procedures."

Exonuclease (dsDNA)b

ss, single-stranded; ds, double-stranded.

complex was judged pure enough to warrant characterization after it had passed through one ammonium sulfate fraction- ation and nine chromatography resins ("Experimental Pro- cedures"). At this point, the purest enzyme (Fraction VIII-A (peak), Table I) had a specific activity of 200,000 units/mg protein on poly(dC), a 53,000-fold increase over the starting cell extract. Furthermore, the ratio of primase activity on poly(dC) compared with its activity on poly(dT) was the same in Fractions VI-A to VIII-A. Similarly, the ratio of primase activity on poly(dC) to DNA polymerase activity on activated DNA was also constant. The final product was free of ATPase, exonuclease, and, endonuclease activities (Table 11). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1) of Fraction VIII-A (peak) revealed a cluster of large polypeptides (Mr = 176,000, 158,000, 133,000, and 118,000) and at least four smaller polypeptides (Mr = 62,000, 57,000, 53,000, and 30,000). This was similar to the composition of KB cell DNA primase-DNA polymerase a purified by immunoaffinity chro- matography (13).

DNA synthesis on poly(dC) or poly(dT) was completely dependent on the presence of GTP or ATP. However, synthe- sis on single-stranded circular M13 virion DNA was inhibited only 66% in the absence of all four rNTPs. Synthesis was inhibited 52% in the absence of ATP alone but only 14% in

I dye front I FIG. 1. Polypeptides in purified DNA primase-DNA polym-

erase (Y (Fraction VIII-A, peak; Table I). A 10-pl sample contain- ing 17.4 units of DNA primase activity and 20.9 units of DNA polymerase (Y activity was subjected to sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (54), and the polypeptides detected by silver staining (55) . Protein standards were run in parallel (rabbit muscle myosin (200,000); rabbit muscle phosphorylase b (92,500); bovine serum albumin (68,000); ovalbumin (45,000); bovine erythro- cyte carbonic anhydrase (31,000)). A densitometer tracing was ob- tained from a photographic negative of the gel using a Joyce Loebk microdensitometer. The molecular weights of major polypqptides are indicated.

the absence of GTP, CTP, and UTP. Apparently, the M13 DNA was either contaminated by oligonucleotide primers and/or broken molecules that could provide primers by form- ing hairpin loops. DNA synthesis on all three templates was completely resistant to 200 pg/ml of a-amanitin, a level sufficient to inhibit RNA polymerases I1 and I11 (34). The DNA polymerase activity that co-purified with DNA primase activity was identified as a-polymerase, consistent with a DNA primase-DNA polymerase a complex as reported for other systems (see Introduction). DNA polymerase activity was inhibited by aphidicolin when assayed on activated DNA, but was insensitive to ddTTP (3). In addition, its chromato- graphic behavior on DEAE-Sephacel, Sepharose CL-GB, and

6256 DNA Primase-DNA Polymerase cy from Simian Cells

r A B

0 5 IO 15 20 25

Elution Volume (ml)

FIG. 2. Separation of DNA primase-DNA polymerase a from DNA polymerase a. Fraction V, containing 11,000 units of DNA polymerase a activity and 2640 units of DNA primase activity, was chromatographed on DEAE-Bio-Gel as described under “Exper- imental Procedures.” Fractions (0.6 ml) were collected and 2-p1 ali- quots assayed for DNA primase (closed circles, poly(dC) template) and DNA polymerase a (open circles) activities for 15 min at 30 “C (“Experimental Procedures”). Fractions under brackets A and B were pooled as indicated and designated as Fraction VI-A and VI-B. Protein was detected by absorbance at 280 nm and the KC1 concen- tration was determined from the refractive index.

hydroxylapatite was characteristic of DNA polymerase a (3, 32, 33). DNA polymerase a stimulatory cofactors C,C, (3, 4) were absent. The K, values for activated DNA and M13 DNA containing two DNA primers/molecule were 48 and 67 pg/ml, respectively (data not shown), the same as observed for CV-1 a-polymerase alone on these templates (4).

Three properties of DNA primase activity were revealed during its purification. First, the purified enzyme preferred poly(dC) template 10-fold over poly(dT) template (Table I). Second, after chromatography on DEAE-Bio-Gel (Fraction VI-A), total units of primase activity increased 1.5-fold, sug- gesting the removal of an inhibitory factor(s). Third, only a fraction of the total a-polymerase activity co-purified with DNA primase. DNA polymerase a not associated with the primase-polymerase complex was completely removed by chromatography on DEAE-Bio-Gel (Fig. 2).

Determining the Exact Size of RNA Primers”T4 DNA polymerase 3‘-5‘ exonuclease (T4 exo) has been used to isolate RNA primers from nascent DNA chains (35). Under appropriate conditions, this enzyme can degrade RNA-primed DNA from its 3‘-end until it encounters the RNA-p-DNA junction. Therefore, [5’-32p]pA(pA)9(pdA)15-30 was incubated with T4 exo for varying periods of time to determine optimal conditions for leaving a single dNMP remaining at the 3’- end of each RNA primer (Fig. 3). After 1 h of digestion, 35% of the [5’-32P](pA)lo(pdA)15-30 was converted into [5’-32P]- (pA),,(pdA). After 2 h, 80% of the radioactivity was 11 nu- cleotides long, and 7% was 10 nucleotides long. Continued incubation for up to 10 h resulted in only two major bands, an 11-mer (42%) and a 10-mer (25%). Oligomers shorter than 10 nucleotides (33%) were evenly represented in the range from 1 to 9 nucleotides. The absence of significant radioactiv- ity in the region from 12 to 40 bases demonstrated that T4 exo digested DNA in a highly processive fashion. Thus, T4 exo rapidly degraded nascent DNA chains, leaving first 1 and then no dNMP residues covalently linked to the 3’-ends of RNA primers.

The Length of RNA and DNA Synthesized on a poly(dT) Template-DNA was synthesized on poly(dT) by DNA pri- mase-DNA polymerase a (Fraction VIII, Table I) in the presence of [cI-~’P]ATP and dATP. Alkaline hydrolysis of the

130

:1 1 1

I

t

1

‘5

.1

I ATP

FIG. 3. T4 exonuclease digestion of a RNA-p-DNA model fragment. [5’-32P]pA(pA)s(pdA)1530 was prepared as described un- der “Experimental Procedures” and digested with T4 DNA polymer- ase 3 ’ 4 ’ exonuclease (T4 exo) for times ranging from 0 to 10 h. Lane a contains [5’-32P]p(Ap)n generated from partial alkaline hydrolysis (56). The ATP was unreacted [y3*P]ATP from the polynucleotide kinase reaction. Lune b contains [5’-32P]pAlo. Numbers on the vertical axis indicate position of 2’(3’)p(Ap)”; the slower migrating band at each nucleotide position is cyclic 2’(3’)p(Ap), (57).

32P-labeled polynucleotide products converted all the label into mononucleotide (Fig. 4, lane lz), demonstrating that only the RNA primers were labeled. Digestion with T4 exo released more than 75% of the radiolabel as an octanucleotide (Fig. 4, lanes i and j ) . Since this material contained a single 3’-dAMP residue, the size of the RNA primer was 7 bases. A small amount of label appeared in the dinucleotide region, but this was resistant to alkali and also present in the undigested (Fig. 4, lane f ) and minus a-polymerase (Fig. 4, lane e) samples, demonstrating that it was a minor contaminant in the radiois- otope. Some label (7%) also appeared as mononucleotides

DNA Primase-DNA Polymerase a from Simian Cells 6257

:A(~A), No Primer

ATP n

a exo T4 A

30 25 25* 20 20*

15 15*

t

a b c d t

t

e f g h i j k FIG. 4. DNA and RNA products synthesized on a poly(dT)

template. DNA primase-DNA polymerase CY (01) (8 units of primase, Fraction VIII-A) was incubated for 1 h at 30 "C with 2.5 pg of poly(dT), dATP, and [m3'P]ATP as described under "Experimental Procedures." Products (No Primer) were divided into 4 aliquots and left untreated ( l a n e f ) , treated with T4 exonuclease (T4 exo) for 3 h (lane i) or 5 h ( l a n e j ) , or treated with alkali (A) ( l a n e k). No products were observed when DNA primase-DNA polymerase a was omitted from the reaction (lane e). Alternatively, products were synthesized with 2.5 pg/ml of [5'-32P]rAIo primer (*pA(pA),) in place of [ c Y - ~ ~ P ] ATP either in the absence (lane a ) or presence (lane b) of 2 mM non- radioactive ATP. Samples were then analyzed by gel electrophoresis in urea. [5'-3'P]p(Ap), (lanes c and h) and [5'-3'P](pA)l~ (lanes d and g) standards were run in parallel. Sizes of standards in nucleotides are indicated on the vertical axis.

which resulted f'm degradation of a minor fraction of primers. Thus, the major product of DNA primase on poly(dT) template in a coupled reaction with a-polymerase was (pp)pA(~A)~. The presence of a 5'-terminal di- or tri- phosphate was confirmed by the ability of these polynucleo-

TABLE I11 Effect of rAlo and ATP on the rate of DNA synthesis by DNA

primae-DNA polymerase a! Reaction conditions"

ATP rA,,

mM

DNA synthesis

pmol dAMP incorporatedl10 min

2 0 2 2 1.25 126 2 2.5 171 2 5 245 2 10 293 2 20 300 0 2.5 169 1 2.5 166 2 2.5 170 3 2.5 174 4 2.5 167

a Reaction conditions are the same as in Fig. 4, except that ['HI dATP (500 cpm/pmol) was the only radiolabeled component, and 0.04 units (poly(dC) template assay) of DNA primase-DNA polym- erase 01 were used.

tides to be labeled in the capping reaction (see below). The DNA products produced by DNA primase-DNA polym-

erase a on a poly(dT) template, with and without a preformed RNA primer, were fractionated by gel electrophoresis to de- termine their length. When provided with [5'-32P]pAlo, the primase-polymerase complex extended this RNA primer with 10-15 residues of dAMP (Fig. 4. lane a). This degree of processivity is characteristic of DNA polymerase a analyzed under a variety of conditions (4, 36, 59-61). All of the RNA primer was utilized in this reaction without the enzyme reini- tiating on the 3'-ends of nascent DNA chains, because a- polymerase prefers RNA primers 200-fold over DNA primers (4, 36). Addition of 2 mM ATP to allow de nouo RNA primer synthesis had no effect on processivity (Fig. 4, lune b) . Under the same reaction conditions, but in the absence of a pre- formed primer and in the presence of [cx-~'P]ATP, nascent DNA chains 22-28 nucleotides long accumulated (Fig. 4, lune f). Since they contained a 7-base RNA primer, processivity was similar to that observed with a preformed primer. Thus, the processivity of DNA primase-DNA polymerase a was unaffected by the source of its primer.

DNA primase-DNA polymerase a prefers to initiate DNA synthesis on pre-existing primers rather than initiate synthe- sis of an RNA primer de nouo. First, the same fraction of [5'- 32P]pAlo was utilized in the presence or absence of ATP (Fig. 4, lunes a and b). Second, under the same reaction conditions, incorporation of [3H]dATP in DNA in the presence of 2 mM ATP was proportional to the concentration of PAlo added, whereas the rate of DNA synthesis in the presence of 2.5 pg/ ml of PAlo was unchanged by addition of ATP (Table 111). Finally, in the absence of a preformed RNA primer, about 35% of the nascent DNA chains were significantly longer than 22-28 bases and accumulated at the top of a 22% gel (Fig. 4, lune f). These chains represented reinitiation of DNA synthesis on the 3'-ends of nascent DNA chains. This was not observed when preformed RNA primers were present because of a-polymerase's strong preference for RNA primers over DNA primers (4,36). The Lengths of RNA Primers Synthesized on a Natural

DNA Template-Single-stranded circular M13 virion DNA was chosen as a natural DNA template to avoid the presence of primers formed from 3' hairpins. However, DNA synthesis on this template was not completely dependent on addition of rNTPs. Therefore, nascent DNA initiated de nouo by DNA

6258 DNA Primae-DNA Polymerase (Y from Simian Cells

G h N ) n PN(PN)n x

I Nuclease -

GPPPU(

GPPPC{

GPPPA {

GPPPG {

GTP{

: )UMP

1 . }CMP

a b c d e f g

a z:o T 2 P1 - “I + - 2 4 1 5 1 5

h i j k l m n o p q r s t

-50

-20

21 5 4 * :lo . . . . -5 . . . -1

-GTP

-ATP . CTP * UTP

FIG. 5. DNA and RNA products synthesized on a M13mp7 DNA template. DNA primase-DNA polym- erase a (8 units of primase, Fraction VIII-A) was incubated for 20 min at 30 “C with M13mp7 DNA, the four rNTPs and the four dNTPs as described under “Experimental Procedures.” The 5’-ends of nascent DNA chains containingpprN or ppprN were “capped” with [cY-~’P]GTP using vaccinia guanylyltransferase. The capped products were divided into four portions: Untreated (a+, lanes a and i); T4 exonuclease (T4 exo) treated for 2 h ( l a n e m) or 4 h ( l a n e n); RNase T2 (T2) treated for 1 h (lanes b and 0) or 5 h (lanes c andp), and nuclease P1 (Pl) treated for 1 h (lanes d and q) or 5 h (lunes e and r ) . Samples were then analyzed either by electrophoresis in polyacrylamide gels containing urea (right panel, lanes h-t) or by chromatography on PEI-cellulose (left panel, lanes a-e). Alternatively, the 5’-ends of polynucleotides synthesized by DNA primase-DNA polymerase CY were labeled with 32P using [T-~~PIATP and polynucleotide kinase. This sample and a sample which had been labeled by the capping protocol were treated with T4 exo and fractionated by electrophoresis in a polyacrylamide urea gel. Material corresponding to full-length primers was isolated from the gel, digested for 2 h with nuclease P1 and chromato- graphed on PEI-cellulose. Lane f shows material which had been labeled by the cappng protocol and lane g shows material which had been labeled by the kinasing protocol. Controls and Standards: capped material from a reaction incubated without DNA primase-DNA polymerase a (a-, lanej ) . SV40 [5’-32P]DNA DdeI fragments ( l a n e h). [5’- 3’P]p(Ap), standards (lanes l and s). [5’-32P](pA)lo standard (lanes k and t ) . Numbers on the vertical axis indicate nucleotide positions of p(Ap)n standards. The positions of GTP, ATP, CTP, and UTP, applied to parallel lanes of the same gel, are indicated. Similarly, UMP, CMP, AMP, and GMP indicate the position of 5’-UMP, 5‘-CMP, 5’-AMP, and 5’-GMP, respectively, on PEI-cellulose, while GpppU, GpppC, GpppA, and GpppG indicate the positions of cap standards.

primase was radiolabeled selectively using vaccinia virus guanylyltransferase to add [ CI-~’P]GTP to polynucleotide chains containing either 5’-pprN or ppprN, generating a 32P- labeled “cap,” G*ppprN(pN), (28). The requirement for a 5’- terminal di- or triphosphate was confirmed by the absence of labeling after treating the substrate with bacterial alkaline phosphatase, and by the inability of single-stranded DNA restriction fragments to act as substrates (data not shown). DNA primase-DNA polymerase a was incubated with M13 DNA in the presence of all four complementary rNTPs and dNTPs, and the polynucleotide reaction products labeled by capping. Since the material was end-labeled, the amount of radiolabel was proportional to the number of molecules. La- beled molecules were 30 residues or longer (Fig. 5, lane i).

After a 2-h incubation with T4 exo, 71% of the 32P-oligonu- cleotides were 9 to 11 residues in length (Fig. 5, lanes m and n). The length of the primer was corrected for the presence of a cap structure by digesting a sample with nuclease P1 to release G*pppN (29). The cap structure migrated at the po- sition of ~ ( A P ) ~ (Fig. 5, lanes q and r ) . In addition, G*pppA(~A)~pdA, the T4 exo digestion product from capped RNA primers synthesized on poly(dT), migrated at the posi- tion of p ( A ~ ) ~ ~ , 3 consistent with a previous report (9). ‘There- fore, the actual size of RNA primers synthesized on M13 DNA was 6-8; 2 residues less than the measured value because of

3Yamaguchi, M., Hendrickson E. A., and DePamphilis M. L., (1985) Mol. Cell. Biol., in press.

DNA Primase-DNA Polymerase (Y from Simian Cells 6259

TABLE IV raphy on PEI-cellulose together with cap standards. Only Effect of ribonucleotide concentration of RNA primer synthesis GpppA (86%) and GpppG (14%) were detected (Fig. 5, lanes

Ribonucleotide d and e). Capped RNA primers were released by T4 exo emphasized digestion. Those corresponding in size from p(Ap), to p(Aphl

A G C U PI, and the products fractionated by chromatography on PEI- in assay were isolated by gel electrophoresis, digested with nuclease

cellulose (Fig. 5, lane f ) . Again, only GpppA (84%) and GpppG Ribonucleotide concentrations in assay (mM)O

ATP GTP CTP UTP

DNA synthesis

5”Terminal base ( % ) d

5’-(p)pprN(pN), (%)‘

(P)PPA (P)PPG (P)PPC (P)PPU

AMP GMP CMP UMP

3”Terminal base (%)‘

RNA primer length distribu- tion (%)f

1-3 4-6 7-9

2 2 2 2 0.2 4 0.2 0.2 0.2 0.2 4 0.2 0.2 0.2 0.2 4

100 116 160 172 100 83 115 134

81 51 86 86 19 49 14 14 0 0 0 0 0 0 0 0

23 20 19 22 49 53 41 47

9 10 18 2 19 17 22 29

10 9 26 16 20 22 23 24 70 69 51 60

See M13mp7 template conditions under “Experimental Proce- dures.”

bIncorporation of total [3H]dNTPs was measured after passing through Bio-Gel P-60 and prior to capping reaction. 100% = 800 pmol of dNMP.

e Incorporation of [a-32P]GTP via the capping reaction was calcu- lated from the radioactivity of RNA primers following gel electropho- resis (e.g. Fig. 6). 100% = 175 fmol of GMP.

Radioactivity in GpppA, GpppG, GpppC, and GpppU was mea- sured by cutting out the appropriate spots from PEI-cellulose (e.g. Fig. 6).

e Transfer of 32P from nascent DNA synthesized with all four [a- “PIdNTPs (0.25 mCi each) to the 5’-ribonucleotide at RNA-p-DNA covalent linkages was measured following hydrolysis in 0.3 M NaOH, 1 mM EDTA for 20 h at 37 “C (18). Prior to hydrolysis, DNA was separated from unincorporated nucleotides by two cycles of gel filtra- tion on Sepharose CL-4B. 32P-ribonucleotides were fractionated by chromatography on PEI-cellulose using 1.6 M LiCl with 2’(3’)-NMP standards run in parallel. Both hydrolyzed and unhydrolyzed samples were chromatographed and NMP spots excised from both lanes in order to subtract any background. 100% = 176 fmol ( A ) , 211 fmol (G), 214 fmol ( C ) , and 173 fmol (v).

’The size of RNA primers in Fig. 6 was corrected by subtracting two nucleotides for the cap structure and one nucleotide for the 3’ - terminal dNMP.

the cap structure and 1 less because of the single 3’-dNMP. RNA primers shorter than 6 nucleotides (the region between p(Ap), and p(Ap), in Fig. 5, lanes m and n) accounted for about 29% of the radio-label. This was more than twice as much as observed with T4 exo digestion of a model substrate (Fig. 3). Hence, DNA primase appeared to synthesize primers 1-5 residues as well as 6-8 residues on a natural DNA tem- plate. The bands in the mononucleotide region (Fig. 5, lanes m and n) which were also present in the sample before T4 exo digestion, migrated like [a-32P]GTP run in parallel, sug- gesting that they were left over [a-32P]GTP from the capping reaction.

The 5 ’-Terminal Ribonucleotides of R N A Primers Synthe- sized on M13 DNA-The 5”terminal ribonucleotides of RNA primers synthesized on M13 DNA template were identified by digesting the total population of polynucleotides with nuclease PI and then subjecting the products to chromatog-

(16%) were detected in the same relative amounts observed with the total sample, indicating that the 5’-ends of full- length primers were the same as those of smaller primers. The presence of a 5’-cap was confirmed by digestion with RNase T2 which yielded products that migrated faster than nuclease P1 products during gel electrophoresis (Fig. 5, lanes o and p ) and slower than nuclease P1 products on PEI- cellulose (Fig. 5, lanes b and c), consistent with the expected structure, GpppNp (38).

Although capping enzyme can react with all four terminal ribonucleotides (39), some preference for A and G may exist under the conditions we used. Therefore, the 5’-ends of nas- cent polynucleotides were treated with alkaline phosphatase and then radiolabeled with [ Y - ~ ~ P ] A T P using T4 polynucleo- tide kinase. Polynucleotide kinase has no preference for par- ticular 5’ termini (40), and it almost quantitatively labels 5’- hydroxyl ends of nucleic acids under these conditions (41). The sample was then digested with T4 exo, and fractionated by gel electrophoresis. Full-length RNA primers migrating in the region between p(Ap), and p(Ap)g were isolated, digested with nuclease P1, and the products fractionated by PEI- cellulose chromatography (Fig. 5, laneg). Only 5’-AMP (86%) and 5’-GMP (14%) were detected their ratio was the same as observed with capped products. Therefore, RNA primers synthesized on natural DNA under the assay conditions em- ployed begin with either ATP or GTP in the ratio of 4:l.

Effects of the Relative Concentrations of Ribonucleoside Tri- phosphates-The strong preference for ATP and secondarily for GTP as initiating nucleotides for primer synthesis presum- ably reflects a preference for certain initiation sites in natural DNA templates. Is this preference influenced by the relative concentrations of the four rNTPs in the reaction mixture? To answer this question, DNA primase-DNA polymerase was incubated with M13 DNA under four different conditions, each emphasizing a preference for one of the ribonucleotides (Table IV). Subsequent incubation of these samples in alkali or with nuclease P1 confirmed that all of the 32P-labeled oligomers were 5’-capped RNA primers (Fig. 6, lanes s-2). A 20-fold increase in GTP concentration increased the rate of DNA synthesis and decreased the rate of primer initiation by 15-20% (Table IV). The most significant change, however, was the ratio of A:G starts which decreased from 4:l to 1:1 (Fig. 6, lanes e and f ; Table IV). In contrast, elevating the concentration of either CTP or UTP by 20-fold increased the rate of DNA synthesis 60-70% and increased the frequency of initiation events 15-35%, but had no effect on the selection of an initiating nucleotide. The ratio of A G starts remained 4:1, and initiation with either CTP or UTP was still not detected (Fig. 6, lanes g and h; Table IV). Therefore, the selection of a rNTP to initiate primer synthesis is restricted to a purine, but the choice between A or G depends on the relative concentration of ATP and GTP in the reaction.

Increasing the concentration of a particular rNTP margin- ally increased its representation at RNA-p-DNA linkages, as measured by the transfer of 32P from DNA to 2’(3’)-rNMPs following alkaline hydrolysis (Table IV). Most notable was the fact that rGMP was present in 41-53% of RNA-p-DNA linkages, regardless of the relative rNTP concentrations. However, increasing CTP or UTP increased the fraction of

6260

short (1-3

DNA Primase-DNA Polymerase a from Simian Cells

N u c l e a s e PI T 4 e x o OH- P1 I I - + - I “

- + ‘“ A G C U ’ A G C U A G C U A G C U A G C U A G C U

Orb a -4: 7,- ”, ?7., i ..n. ,”I .F.-y 1 Ori

.lo

6 5

.l

FIG. 6. Effect of rNTP concentration on size and composition of RNA primers. DNA primase-DNA polymerase a (8 units of primase, Fraction VIII-A) was incubated with M13mp7 DNA as described in the legend to Fig. 5, except that reactions contained different rNTP concentrations. Reactions are designated A, G, C, and U. A, 2 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 0.2 mM UTP. G, 2 mM ATP, 4 mM GTP, 0.2 mM CTP, 0.2 mM UTP. C, 2 mM ATP, 0.2 mM GTP, 4 mM CTP, 0.2 mM UTP. U, 2 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 4 mM UTP. The 5’-ends of nascent polynucleotides were capped, divided into four portions, treated as described below, and fractionated either by chromatography on PEI-cellulose (lanes a-h) or by electrophoresis in a polyacrylamide urea gel (lanes i-2). Untreated (lanes a-d; A ) . Nuclease P1 (Pl) treated for 2 h (lanes e-h; w-2). T4 exonuclease (T4 exo) treated for 2 h (lanes 0-r). Alkali treated (OH-, lanes s-0). Lune rn contains [5’-32P](pA)lo and lane n contains [5’-3zP]p(Ap),. Numbers on the vertical axis designate the nucleotide positions of p(Ap)n standards.

residues) primers at least 2-fold, indicating that the transition from RNA to DNA synthesis occurred more quickly with pyrimidine-rich primers than with purine-rich primers. The lengths of RNA primers in the G-rich reaction were indistinguishable from those in the A-rich reaction (Fig. 6, lanes o and p ) .

DISCUSSION

The length of RNA primers synthesized by enzymes puri- fied from other sources is difficult to compare with our results, because DNase I was used to release the RNA primer from nascent DNA. DNase I leaves a variable number of dNMP residues attached to the 3’-end of the primer, estimated to be between 1 and 3 (42), whereas T4 exo left a single 3”terminal dNMP (Fig. 3). Therefore, to estimate the size of the ribo- oligonucleotide portion in published work, we subtracted 1 residue from the longest oligonucleotide and 3 from the short- est oligonucleotide to reflect the variation in 3’-dNMP resi- dues. Thus, the size range for RNA primers synthesized on homopolymer templates was 5-14 residues: poly(dT), 5-14 (10, 15); poly(dC), 7-14 (18); poly(dIT), 7-9 (6). Similarly, the size range for RNA primers synthesized on natural DNA templates was 4-14 residues: 12-14 for Drosophila primase (15), 4-13 for human primase (13), 6-10 for mouse primase (43), and 5-9 for yeast primase (19). Thus, the average size of RNA primers measured in this manner is 7-12 bases. How-

ever, when both T4 exo and DNase I were used to release RNA primers synthesized on a natural DNA template added to a Drosophila embryo extract, the primer was shown to be (p)ppApA(prN)r-5 (9), in excellent agreement with the results for CV-1 cell DNA primase-DNA polymerase a showing a 7- base primer on poly(dT) (Fig. 4), and a 6-8-base primer on M13 DNA (Fig. 6). Thus, it appears that variation in the length of RNA primers synthesized in vitro reflects a variation inherent in the DNase I procedure, and not in the source of DNA primase.

The average size of RNA primers synthesized in vitro appears shorter than the size reported in vivo. SV40 DNA replicating in CV-1 cells contains RNA primers with an average length of 9-11 bases, as measured by the T4 exo method (27). This is 3 bases longer than observed with CV-1 DNA primase-DNA polymerase a. Similar experiments re- vealed that RNA primers synthesized in Drosophila embryos were 8 bases (44), 1-2 bases longer than observed in vitro (9). In both examples, RNA primers synthesized in vivo or in vitro contained 70-80% 5’-(p)pprA and 20-30% 5’-(p)pprG.

DNA primase-DNA polymerase a strongly preferred to use pre-existing 3’-OH termini as primers rather than synthesize new RNA primers de novo (Fig. 4). Thus, this enzyme would not initiate DNA synthesis on forward arms of replication forks, where the direction of DNA synthesis is the same as the direction of fork movement, because the nascent DNA

DNA Primase-DNA Polymerase a from Simian Cells 6261

chains provide pre-existing primers. In fact, 99% of the in vivo RNA-primed initiation events in SV40 replicating DNA molecules occur on retrograde arms of replication forks (27), although the situation for polyoma virus is less clear (2). I t seems likely that this preference is a property of the primase- polymerase complex because it was highly purified away from a-polymerase (Fig. 1; Tables I and 11). However, if DNA primase activity is less stable than DNA polymerase a activity (18), purified preparations may still contain mixtures of pri- mase-polymerase complex and polymerase alone.

DNA primase-DNA polymerase a first produced RNA- DNA chain 20-25 nucleotides long which were then extended by further DNA synthesis (Fig. 4, lane f ) . This could explain the accumulation of small RNA-primed DNA chains in polyoma replicating DNA (58). Furthermore, DNA polymer- ase activity coupled to DNA primase activity is not inhibited by aphidicolin (a specific inhibitor of a-polymerase (62)), while the same enzyme preparations using preformed primers are inhibited by this drug (data not shown; 6, 13, 19). Thus, reinitiation of DNA synthesis on short RNA-DNA chains could explain the fact that all steps in DNA synthesis at replication forks in cellular, SV40 and polyoma virus chro- mosomes are inhibited by aphidicolin (1, 2, 62).

Although it has been shown that the ratio of dNTPs to rNTPs in the reaction affect the base composition and size of RNA primers (12, 45-47), the affect of changing the relative concentration of each rNTP had not been examined. When purified DNA primase-DNA polymerase a was incubated with rNTPs alone to prevent DNA synthesis, the size of the RNA synthesized increased 2-3-fold (11, 47). Similarly, when iso- lated nuclei were incubated with high ratios of dNTPs to rNTPs (45, 46), dNMPs were misincorporated into the RNA primer, producing a primer of mixed composition and result- ing in the release of shorter primers upon digestion with DNase I (45). When purified CV-1 DNA primase-DNA po- lymerase a was incubated with varying concentrations of ATP and GTP, the size of RNA primers remained the same but the 5”terminal nucleotide composition changed dramatically. The ratio of 5’-ATP to 5’-GTP decreased from 4:l to 1:l as the relative concentration of GTP/ATP was increased 20-fold (Fig. 6; Table IV). A similar result was observed during the replication of polyoma DNA in isolated nuclei (58). High concentrations of CTP or UTP had no effect on the choice of the initiating rNTP, but the proportion of shorter primers (1-3 bases) increased %fold (Table IV). From the above results, we conclude that DNA primase always initiates at sites beginning with either dT or dC in the template, and the frequency a t which a particular site is used depends on the relative concentrations of rATP and rGTP. The fact that 60- 73% of the RNA-p-DNA linkages contained rA or rG, regard- less of the concentration of CTP and UTP (Table IV), dem- onstrates a strong preference for purines at 3‘-ends of RNA primers. However, increasing the concentration of CTP or UTP did increase slightly the frequency of a pyrimidine at RNA-p-DNA junctions with a concomitant increase in the fraction of shorter RNA primers, suggesting that purines in the template can promote premature termination of RNA synthesis. This model could explain the previous observation that omission of rCTP during DNA synthesis in isolated nuclei had the most dramatic affect on nearest-neighbor fre- quencies (46).

ATP concentration in mammalian cells is on the order of 1-2 mM (48,49). This is 5-10 times greater than other rNTPs (48-50) and approximately 100 times greater than dNTPs (48,49,51,52). Therefore, the standard assay conditions used in this paper are similar to in vivo nucleoside triphosphate

concentrations. However, if precursors for DNA and RNA synthesis are “channeled to replication forks in mammalian cells (53), then local concentrations of rNTPs or dNTPs may vary at different stages in replication and thus modulate RNA primer synthesis in vivo through the selection of initiation sites?

Acknowledgments-CV-1 cells were grown at the Massachusetts Institute of Technology Cell Culture Center, Cambridge, MA. We thank Drs. Earl Baril and Marc Charette for their advice in purifying DNA primase-polymerase a.

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DNA Primme-DNA Polymerme a from Simian Cells 6263

SUPPLEMENTARY MTERIAL To

DNA PRIME-DNA POLYMERASE a FROM SlnIm CELLS: nodulation of RNA primer Synthesis by Ribonucleoside Triphosphates

n. Yanaguchi. E.A. Bendrickson. and 11.L. DePamphilis

EXPERIMENTAL PROCEDURES

" - ~ "~ . . ~ ~ ~ ~ bacterial alkaline phosphatiie-F frbm Wortbington Biochemicals. nuclease PI from Boehringer-Mannbein. and vaccinia guanylyltransferase and RNase Tl fron Bethesda Research Laboratories. Unlabeled nucleotides. homopolymers and poly(rI)-agarose (type 6) were purchased from P-L Biochemicals. radioactive nucleotides from New England Nuclear. o-ananitin from Bnchringer-aannbeim. phenylrthysulfonyl

DEAE-cellUlo8e paper (DESI) from Whatman. DEAE-Sephacel. Sepharost fluoride and leupeptin from Signa, DEAE-cellulose (DE51) and

CL-48. SepharDSe CL-60. and CNBr-activated StphllrOBe from Phsmacia. DUE-Biogel. Biogel P-60 and Silver stain Kit from Bio-Rad. Butyl-agarose from Miles-Yeda. hydroxylapatite (Bypatrite-C) from Clarkson. and polyethyleneinine (PBI) cellulose from BrinkMnn. Single-stranded 1113mpl virion DNA (7231 Dufleotidesl and it8 double-stranded replicative form were prepared as described ( 2 1 ) . ss was double-stranded and single-stranded DNA-Sepharaee (3). CV-1 cells were grown at the Massachusetts Institute of Technology Cell Culture center.

avffus - Unless otherwise specified, all buffers used for Purification of DNA primase-DNA polprase a contained 1.11 phenylrtbyleulfonyl fluoride, 10.11 sodium metabisulfite and 1pgI.L leupeptin to inhlbit proteases. Buffer A is J O m Tris-BCl (pB 1.1). 0.5.11 dithiothreitol, 0.lM EDTA, and lS% glycerol. Buffer B is 10.11 K p O 4 (pB 7 . 1 ) , 1.11 dithiothreitol. 0.1.11 EDTA. and 15s glycerol. Buffer C is 5011 K O 4 (PB 1.6), 400.11 KCl, 1.11 EDTA. I d ditbiothreitol, and 10% glycerol. Buffer D is SO& Tris-BC1 (pB 7 , s ) . 0.111 KC1. 1.11 EDTA. 1.11 dithiothreitol. and 10% glycerol. Buffer E is SO11 Tris-BC1 (pH 7.5). 1.11 EDTA, and I d dithiothreitol. Buffer F is 50.11 Trim-BC1 (PB 1.51, 0.lm EDTA, I M dithiothreitol. and 20s glycerol.

w e % a % % % % % African Green wnkey Cy-1 cells gram to confluency in Dulbecco's mdified Eagle's medium supplemented with 10% calf serum ( 1 x 1 0 1 cellslroller bottle). The mediun was changed 14-10 h before harvesting to stimulate resting cells into S-phase. Cells were harvested as described (37). except that 1.11 phenylmethylsulfonyl fluoride. 10.11 sodium aetabisulfite and l v g l l f . leu ptin were added to the buffers. All steps were carried out at

- ~ * € I a a W "

l&. DNA primase and DNA polymerase-. activities were extracted from

mixed with 1 0 0 11. Of Buffer A containing 0.111 KC1. broken in a Dounce 2 1 O m L of cell suspension taken from 50 roller bottles. The cells e r e

homogenizer with 10 strokes of pestle B and then sonicated for 30 8 at

15,000 x g for 10 min. and the supernatant filtered through four layers 10 kBr with a Branson sonicator. Thhe homogenate waB centrifuged at

Of cheesecloth and centrifuged for 1 b at 100.000 I g. "he supematant [Fraction I) was adjusted to 0.411 Kc1 by diluting with Buffer A. Nucleic acids were removed by stirring Fraction I slorly with 100- of SOgm of DEAE-CellYlose equilibrated with Buffer A containing 0.411 KCl. After 3 0 mi", the slurry was poured over a bed of 10- of DEAE-cellulose to make a column which was then washed with 3 voluus of the same buffer. The pass-through (Fraction 11, 3YOlf. I was adjusted to

610wlv for 3 0 min and then allowed to stand for 60 mi". The SJS saturation with mlid ammoniun sulfate. and the mixture stirred

precipitate was collected by centrifugatirn at 15,000 a 9 for 4J min. dissolved in 30- of Buffer 0, dialyzed against 133 vclurll of Buffer B containina 10.11 KC1. and finallv aDDlied to a DEAE-Sephacel colum (1.5 x II.5cm)~eq~ilibrated with the-.& buffer. After wishing with 3 volumes of the same buffer, the protein was eluted with a 400- linear gradient of l o w to 4 5 O m KC1 in Buffer 0. Bath DNA prinse and DNA polymerase a activities eluted a~ a single peak between 170.11 and 310111 KC1 (16mL). Protein was precipitated with 66s saturated a m n i u n sulfate and dissolved in ImL of Buffer C (Fraction 111).

equilibrated with Buffer C and eluted with the same buffer. DNA polymerase a activity eluted as a broad peak with an apparent molecular weight of 60.000-540.000. DNA primase activity (Fraction IV. 31lf . j eluted at the front of this peak with an apparent molecular weight of 2JO.000-540.000. Molecular weight standards were blue dextran 1000

Fraction I11 was applied to a Sepharose CL-60 column (1.5 I31cm)

(void volume); bovine catalase ?lSO,OOO); bovine serum albumin (61.000); horse cytochrom C (12.500).

equilibrated with Buffer C. The column wrm washed with 2 volurs of Buffer C. and then developed with a 70- linear gradient of 5.11 K P O q

DNA polymerase . activity eluted as a broad peak between 44.11 and 91.11 K P O while DNA primase activity eluted in the center of this peak betken 11.11 and 16.11 KPO,. Protein in the DNA PriMSe peak (17lf.) was precipitated with 66% Batmated a-nium sulfate. dissolved in llf. of Buffer D (fraction VI and stored at - lOOC.

Fraction Iv was applied to a hydroxylapatite coluvl (1.0 x 1 0 . J ~ )

(pB 1.6) 100011 KC1 to 150.11 KPD4 (pB 1.6) + JOO.11 KC1 in Buffer C.

Fraction V from 100 roller bottles was dialyzed for 10 b aqainst

DEAE-Biogel colunn (0 .1 x 6.Ocm) tquilibrated with the same buffer. IS0 volumes of Buffer B containing 40.ll KC1 and then applied to a

The column was washed with 1 S . L of the same buffer and then developed with a 40- linear gradient of 40.11 to 500.11 KC1 in the same buffer. DNA primase plus 66% of the DNA polymerase . activity was recovered in the flow-through IFraCtion VI-A, Fig. 1 1 . Thhe remaining DNA PolymeraBe activity was eluted with 6J .11 KC1 in the same buffer (Fraction VI-Bi Fig. 1). Protein in Fractions VI-A and VI-B were precipitated by 66% saturation with ammonium sulfate. dissolved in 1.J11. of Buffer D, and stored at -2OoC.

Fraction VI-& and VI-0 were dialyzed for 4 h against 133 volurs of Buffer E containing 111 ammonium sulfate. then applied Beparately to

The column was washed with 511. of the sa= buffer and then DNA primase a butyl-agaI0.e column (0.6 1 l.4cm) equilibrated with the sane buffer.

and DNA polymerase a activities were eluted with 4.5- of Buffer E

VII-0). ChrolatOqraDhV on butvl-aaa<OSe resulted in L 7-fold containing 10s glycerol and 0 . 5 % Triton X-loo (Fractions VII-A and

purification with.. ? S i loss in activity. for 10 h aoainst 3 0 0 volumes of Buffer F containina 0.1111 KCl. then

Fractions VII-A and VII-B (1.511. each) were immediately dialyzed

applied separately to a double-stranded DNA-Sepharosc cO1um (0.6 x

column (0.6 I 3.Scml. The columns were washed with l3IL of the same 3.Scm) which was connected directly to a single-stranded DNA-Sepharose

buffer and the flow-through applied directly to a poly(rI)-agarose

washed with S v0lUmes of the same buffer. and then DNA prinase and DNA column (0.3 x 6cm) equilibrated with the same buffer. The column wae

polymerase activities eluted with J I L of Buffer F containing 0.111 KCl. These steps resulted in an 1-fold purification with no loss of activity

Fraction VIII-0. 0.1.L). and one-third of each stored at - 10°C in (Table I). Active fractions were combined (Fraction VIII-A. 0.9lf.i

small aliquots. and two-thirds dialyzed against Buffer D and stored at

decreased by 113 and polyrrrase activity decreased by 11s. -1OOC for immediate use. After 6 months at -lO°C, primase activity had

-e A88av8 - All enzyme assays were shown to be linear over the time period of the assay. DNA polymerase a was identified by its

sensitivity to apbidicolin, lack of sensitivity to ddTTP. and chromatographic behavior (3).

1 5 - 3 0 min in lOuL containing S O m U Tris-BC1 (pB 1.4 at 30%). 1mU dithiothreitol. IOrgtmL DNase I activated calf thymus DNA (13). 0.ld each of MTP. dGTP. dCTP and 13BldTTP (60c~ml~mol). 15% alvcerol. 400

L) D A P- 5 - DNA polymerase a was assayed at 3OoC for

adsorbed Onto DEAE paper and the amount of radioactivity measured (24). ug1N. bovine serum albumin. 10.11 to 40- Kt1 bnd enzyme.- 6NA was then

One unit of DNA polymerase D was defined as 1 m o l of total dNTPs incorporated per hour at 30OC.

(it) DNA eri.p9+ - DNA primase activity was measured at 30oC for IS-30 mln as rNTP(s)-dependent DNA synthesis an a single-stranded DNA template coupled with DNA polymerase a. With homopolymer templates, ZOeL of reaction mixture contained JO.11 Tris-BC1 IpB 1.0 at 30OC). I O M

glycerol. 1" dithiothreitol. 0.Spg poly(dC) or poly(dT). 4mU GTP or Mg-acetate, S o d l Na-acetate, 1 0 O p g l m L bovine serum albumin, Is%

ATP. 0.1011 13BldGTP 1240 cpmlpnal) 01 0 . l M 13BIMTP (240 cpmlpmol) and enzyme. With single-stranded circular 1113mp7 DNA template. 10rL of reaction mixture contained Somu Bepes (pB 1.1). IOOrglmL bovine serum albumin. 1.11 dithiothreitol, 15% glycerol, 13- 119-acetate, I O d Na-acetate. leg 1113mp7 DNA, 4.11 ATP, 0.2 111 each Of GTP. CTP, and UTP. 0.1.11 each of MTP. dGTP. dCTP, and 13BldmP (240 cpdpocl) and enzyme. DNA was processed as demcribed above for DNA polymerase .. One unit of DNA primase activity was defined as 1 "mol Of total dNTP(s) incoiporated per hair at 30Oc. The concentrations of 119- and Na-acetate were optimized for the standard assays. For the poly(dT) template, the optimum range was 4O-llOM Na-acetate and 4-16mU uncomplexed 119-icetate. For the 1113 template, the optimum range W ~ B 5-30.11 Ra-acetate and 1-6.11 uncomplexed 119-acetate.

out u n z D N A priMse conditions. Randomly broken. 5*-.J2P-labcled.

endo- and exonuclease activities. DNA digestion was assayed am a full-length. single-stranded 1113.~1 DNA was used to assay single-strand

decrease in 31P-1113 linear molecules detected after fractionation by electrophoresis in 1% alkaline agarose gels (25). Ouantitation was done by densitometry of autoradiograms. Double-stranded specific endonuclease activity was r a m r e d as the conversion of superhelical.

double-stranded exonuclease activities were assayed as the circular SV40 DNA into relaxed circular SV40 DNA (3). S"3' and 3"s'

disappearance of 5'-3lP or 3@-31P end-labeled ode I restriction fragments of sV40 DNA. The fragments were separated by electrophoresis

bands was quantitated by densitometry of an autoradiogram. in 12% polyacrylamide containing Ill urea, and the disappearance of

(iii) DeoIyribonuClea~e~ (w - All DNase assa 8 were carried

Ile) -e - ATPase activity was measured as the conversion of f.-3aPlATP to Ia-3aPIADP in the presence or absence of 1 p g single-stranded circular 1113.~7 DNA and the presence of 1.11 A T P (16). Reactions were carried out at 30% for 60 nin under DNA primase assay conditions. Aliquots were then chromatographed on PEI-cellulose together with ADP and ATP standards, ADP Spots were excised and radioactivity determined using a scintillation counter.

Analvsis of DNA and RNA Products Synthesized & DNA PrimBe-DNA Polymerase 5.

DNA priMSe sssays*ni& 1.11 A T P and 1OOrCi of I.-32PlATP were Te late Following 1 1 h incubation at 300C. lOOpL

stopped with 11.11 EDFA and 0.5% SDS, then incubated at 31OC for 30 mi" with 10O~gllf. proteinase IC. DNA wan extracted with phenol and filtered through a 6 I L Biogel P-60 column in 10 Trie-BCl (pB 1.01, 1.11 EDTA, and 10.11 NaC1 to remove ""reacted ~s-~$I&TP. Void fractiona were concentrated against 1-butanol. DNA was precipitated witti 1JS ethanol at -109: for 16 h. collected by centrifugation. and then dissolved in

DNA polymerase 3'5' exonuclease (11 ) in 10-1 containing 50.11 Tris-BC1 l3pL 820. Aliquot6 containing about 1.lpg DNA were digested with T4

polymerase. Prior to addition of T4 DNA polymerase. the reaction (pB 1.0). 6.11 11gC11, 11111 KC1, Irg tRNA and I units of T4 DNA

mixture was denatured at 100% for 1 min and quickly chilled. Digestion was carried out at 3loC for indicated periods. Separate aliquot6 were incubated in lOcL of 0.1111 11.08. I.* EDTA at 31Oc for 1 0 h to hydrolyze RM. All samples were then fractionated by electrophoresis in 11% polyacrylanide gels (0.5m x 33cm x 4 1 ~ ~ ) containing 111 urea. 100.11 Tris-borate (pH 1.3) and 1.11 EDTA ( 1 1 ) . Electrophoresis was carried out for 5-1 h at 1500 v using xylene cyano1 and bromphenol blue as tracking dyes. Gels were then overlaid with Saran Wrap and exposed to Kodat X X - 5 film with a Crone=-plus intensifying screen at -1OOC.

primase asmay containing 1113mp7 DNA was incubated for 2 0 nin at 30%. The reaction was stopped and DNA purified as described for the poly(dT) template. Following Biogel P-60 gel filtration. S'-termini were radiolabeled using either TI polynucleotide kinase and [y-3aPlATP after first removing terminal phoaphate6 with bacterial alkaline phosghatase

to 'cap. J'-(p)pprN termini ( 1 1 ) . The capping reaction was carried out (17). or were labeled with vaccinia guanylyltransferase and [a- lplGTP

in lOcL Containing SO.11 T is-BCl (pH 1.9). l.ZJ.11 ngcl2. 6.ll Kcl. 2.s.11

guanylyltransferase. Prior to addition of enzyme. the reaction mixture dithiothreitol. 10~11 I.-3hlGW, and 6 unitm of vaccinia

was denatured at lOOOC for 1 min and then quickly chilled. After

addition of 1 rL of 0.3N B m A . DNA was separated from unreacted incubation at 31OC for 4 5 .in. the reaction was terminated by the

[--31PlGTP by gel filtration throuqh Biouel P-60. AliquotB of lS'-3aPlpolynucleotides were digeated with T4 DNA polymerase exonuclease. or incubated in NaOB as described above. In addition,

Ns-acetate (pB J.3). 1.9 RNA and leg enzyme for 1 h at 31OC. Products digestion with nuclease P1 ( 1 ) ) was carried out in 1OpL containing 30.11

were separated by chromatography on PEI-cellulose plates ( 4 0 x 1Ocm) using 1.611 lithium chloride for 1-9 h at room temperature. Plates were

with a Crone=-plus inteneifing screen at - 7 O O c . Radioactive epots were then dried. covered with Saran wrap and exposed to Kodak X X - S film

Standards were applied in parallel with the sample and visualized with excised and 31P ratsurcd in a toluene-based scintillation fluid.

(.i.&.l W e - S t r a n d & P 1 3 m 1 DNA T e m n m - A 1OOeL DNA

"lt~.violet light.

polymerase I (Klenow fragment, 0.6 units) in the presence of 0.16011 slowly to Goom temperature. "he-primer was extended by E. coli DNA

d M T P and 0.00111 M T P for 3 0 min (30). The extended material was isolated following electrophoreeis in a 11% polyacrylamide-111 urea gel

slice as described below, except that the extract of polynucleotides 0 1 ) . IJ'-3aPIDNA 15 to 40 nucleotides long was extracted from a gel

wan precipitated three times with lJ% ethanol at -10% instead of adsorbing to DEAE-sepbaccl. and then dissolved in 300.L 810.

aml&&.u 54 ril4.l eriaur frpp ePLvacrvlami& Gcl& - RNA primers separated in a 21% polyacrylamide gel containing 111 urea were extracted from gel slices in 0.JM NE,-acetate. 10111 119-acetate, 0.1% SDS at 60OC

gel. the supernatant was diluted with 10 volumes Of 0.0111 NIIqECO3. and for 16 h. After Centrifugation for S nin at 12.000 I g to remve the

then adsorbed to a 3OOeL DEAE-Sephacel column equilibrated with 0.0111 NB4BCD3. After extensive washing with the same solution. RNA primers were eluted with 111 NB4BCD3. and NB4BCO3 was remved by lyophilization (31). Recovery was 10-10%.