Cell cycle-dependent biosynthesis of Plasmodium falciparum DNA polymerase-α

EXPERIMENTAL

PARASITOLOGY

73,

93-100 (1%)

Cell Cycle-Dependent

Biosynthesis of Plasmodium DNA Polymerase-a

INPYO CHOI AND Department

of Radiation

Oncology,

Medical

Ross B. MIKKELSEN

College of Virginia,

Richmond,

Virginia

232984058

R. B. 1991. Cell Cycle-Dependent Biosynthesis of PlasmoDNA Polymerase-a. Experimental Parasitology 73, 93-100. The DNA dium falciparum polymerase-o of Plasmodium falciparum was characterized according to aphidicolin sensitivity and immunological reactivity with monoclonal anti-sera against human DNA polymerase-a. Two major (105 and 72 kDa) and two minor (180 and 130 kDa) catalytic subunits of P. fulciparum DNA polymerase-a were detected on activity gels. Activity gels did not indicate the presence of a DNA polymerase-8 in P. falciparum. Metabolically labeled polypeptides at 180, 105,72, and 52 kDa were immunoprecipitated from Plasmodium nuclear extracts with the anti-KB cell DNA polymerase-a monoclonal antibody and, by size, correspond to the major subunits of mammalian DNA polymerase-a. The monoclonal antibody also neutralized Plasmodium DNA polymerase activity. Plasmodium DNA polymerase was synthesized predominantly at an early schizont stage at which time the parasite began to synthesize its DNA and multiply. No evidence for phosphorylation of the major catalytic subunit was obtained. Plasmodium growth, DNA synthesis, and DNA polymerase activity were inhibited significantly in parallel by aphidicolin. These results suggest that P. falciparum has a typical eukaryotic DNA polymerase-u and that regulation of its activity appears to be at the transcriptional level. o 19% Academic press, hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Plasmodium falciparum; DNA polymerase; Cell cycle; Aphidicolin; Dithiothreitol (DTT); Polymerase chain reaction (PCR); Phenylmethysulfonyl fluoride (PMSF); 4,6-Diamidino-2-phenol-indole dihydrochloride (DAPI); Sodium dodecyl sulfate (SDS). CHOI,

I.,

falciparum

AND MIKKELSEN,

is associated with the 180-kDa subunit and DNA primase activity is associated with In most eukaryotic cells, there are four the two smaller subunits. DNA polyDNA polymerases (OL,l3, y, and 6). They merase-cwin contrast to other DNA polydiffer in size and subunit structure, inhibi- merases is inhibited by aphidicolin. The level of DNA polymerase-a activity tor sensitivities, their respective roles in oscillates during the cell cycle, but the conDNA replication, and interactions with cell trolling mechanisms that regulate this oscilcycle control elements (reviewed in Camplatory behavior are incompletely underbell 1986; Lehman and Kaguni 1989). stood. There is evidence for the phosphorDNA polymerase-ol (DNA polymerase I ylation of DNA polymerase-a (Donaldson in yeast) in its association with primases is and Gemer 1987; Wong et al. 1986). The responsible for chromosomal DNA replicaincrease in activity of DNA polymerase-a tion (Lehman and Kaguni 1989). Because DNA polymerase-cx is very sensitive in of quiescent human fibroblast when stimuvitro to endogenous proteases, there has lated to divide is correlated with an inbeen some confusion concerning its subunit creased phosphorylation of two subunits structure. It is now recognized that DNA (Cripps-Wolfman ef al. 1989). Phosphorylapolymerase-a is composed of four subunits, tion of HeLa cell DNA polymerase-a ina large subunit of 180 kDa or, more often, a creases its catalytic activity by 2- to 3-fold group of polypeptides (140-M kDa), a 70- (Krauss ef al. 1987). Other studies have kDa subunit, and two smaller polypeptides demonstrated a correlation between de n~vo biosynthesis of DNA polymerase-a (60 and 50 kDa). DNA polymerase activity 93 OO14-4894/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

94

CHOI AND MIKKELSEN

and total cellular polymerase activity. For we report on our studies identifying a P. example, in TC7 monkey and 3T3 mouse fulciparum DNA polymerase-a on activity cells, DNA polymerase-o. activity increases gels and by immunoprecipitation with a difin the S phase corresponding with an in- ferent monoclonal antibody raised against creased synthesis of DNA polymerase-a the human KB cell DNA polymerase-a. (Thommes et al. 1986). The steady-state The synthesis and activity of this polymerlevel of DNA polymerase-ol mRNA in pro- ase are cell cycle dependent and correlate liferating human lung fibroblast cells is 20- with the onset of DNA synthesis. fold greater than quiescent cells (Wong et al. 1988).

The erythrocyte stage of the human malaria parasite Plasmodium falciparum replicates itself IO-20 times during the last 4-6 hr of its 48hr life cycle. Studies with synchronized cultures indicate that DNA synthesis starts at 29.5 to 31 hr after parasite invasion of the erythrocyte and continues through most of the remainder of the life cycle. Both hydroxyurea and aphidicolin are effective inhibitors of P. falciparum DNA synthesis and block late steps in the intraerythrocytic parasite maturation (Inselburg and Banyal 1984), suggesting that a typical eukaryotic polymerase-cy is involved. In a fraction of the erythrocyte stages, the parasite does not undergo asexual development. Rather, these parasites carry out one round of DNA synthesis and subsequently differentiate into gametocytes that mature into male and female gametes and which upon fusion in the gut of the mosquito complete the sexual phase of the parasite life cycle (Janse et al. 1988). The controlling genetic elements that determine whether a parasite is committed to asexual maturation or gametocyte differentiation are not understood but must necessarily involve regulation of DNA polymerase-a activity. Abu-Elheiga et al. (1990) have recently demonstrated a DNA polymerase-a activity in P. falciparum as defined by sensitivity to aphidicolin. The polymerase, according to activity gel analysis or molecular sieve column chromatography, was characterized by a M, of 100-103 kDa and was not inhibited by a monoclonal antibody raised against human DNA polymerase-a. Herein,

MATERIALS

AND METHODS

The T9/96 clone of P. falciparum was cultured by a modification of the Trager-Jenson method in T-75 flasks at a 5% hematocrit as previously described (Choi and Mikkelsen 1990). Where indicated, cultures were synchronized by the Percolhsorbitol method (Aley et al. 1986; Lambros and Vanderberg 1979). Stages of Plasmodium development were classified relative to the time of sorbitol lysis (t = 0) and were verified by Giemsa staining: stage 1 (early rings), O-7 hr; stage 2 (late rings), 7-12 hr; stage 3 (trophozoites), 12-23 hr; stage 4 (early schizont), 23-27 hr; and stage 5 (late schizont), 27-32 hr. Alternatively, parasites at different stages of maturation were obtained from asynchronous cultures by centrifugation on Percoll gradients (Aley ef a/. 1986). Nuclear extracts of P. falciparum cultures were prepared according to the method of Manley er al. (1983). Briefly, parasites free of the erythrocyte membrane were obtained by N, cavitation and were further purified on Percoll density gradients (Choi and Mikkelsen 1990; Read and Mikkelsen 1990). Free parasites were resuspended in four packed cell volumes of 10 mM Tris-HCI, I mM EDTA, 5 mM DTT, and 1 mM PMSF, pH 7.9. After 20 min on ice, parasites were homogenized in a Dounce homogenizer and four packed cell volumes of 50 mM Tris-HCI, 10 n04 MgCI,, 0.5 mM EDTA, 2 mM DTT, 25% sucrose, and 50% glycerol were added followed by dropwise addition of 1 vol of saturated ammonium sulfate. The Iysates were centrifuged at 70,OOOgfor 3 hr and the resulting supematents were dialyzed against 25 mM Hepes, 100 mM KCI, 12 mM MgCl,, 0.5 mM EDTA, 2 mM DTT, 0.5 mM PMSF, and 17% glycerol. Nuclear lysis was monitored by staining with DAPI. Anti-KB cell DNA polymerase-a monoclonal antibodies (SJK-132-20 and SJK-287-38) were gifts from Dr. J. M. Collins (Medical College of Virginia, Richmond, VA). These two monoclonal antibodies were pooled for most of the experiments as described in the original report on the development of the monoclonal antibodies (Wang et al. 1986). The antibodies appear to recognize different epitopes but immunoprecipitate the same set of polypeptides (180, 165, 77, 55, 52, 49, 23-25 kDa) from human cells. SJK-132-20 is approximately twice as effective in neutralizing KB cell DNA

P. fakiparum:

DNA

POLYMERASE-cx

95

polymerase-a activity as SJK-287-38 (Tanaka et al. boiled in Laemmli sample buffer. SDS-polyacrylamide gel electrophoresis was performed as described 1982). The latter antibody used by Abu-Elheiga et al. (1990) was found to be ineffective in neutralizing P. (Laemmli 1970). falciparum DNA polymerase-a activity. Activated calf thymus DNA, pBR 322, and pBR 322 BamHI primers were purchased from Sigma Chemical RESULTS AND DISCUSSION Co. (St. Louis, MO). The DNA polymerase chain reaction (PCR) was done essentially as described by DNA polymerase activities were deMullis and Faloona (1989) except that pBR 322 (0.1 nM) and pBR BamHI were used as clockwise and tected in nuclear extracts of P. falciparum counterclockwise primers (10 nM) for target DNA. Afby using a PCR method (Fig. lA, top ter reaction, 62-bp fragments bounded by the two frame). There was no measurable DNA primers were amplified. polymerase activity in noninfected erythroMeasurements of DNA synthesis, the DNA polymerase activity assay, and activity gels were per- cytes (Fig. IA, bottom frame). With nuclear extracts from infected cells the PCR formed as described previously (Inselburg and Banyal 1984; Karawya and Wilson 1982; Blank et al. 1983). In was saturated after six cycles. At this time, the case of the activity gels, lysates from parasites and the amplified fragments rehybridized more uninfected erythrocytes prepared as described above were adjusted to 65 mM TrisHCl, 1% SDS (BioRad), 1% mercaptothanol, 2 m&f EDTA, and 10% glycerol, incubated for 3 min at 37”C, and immediately loaded onto a 10% polyacrylamide SDS gel. The latter was prepared as described (Laemmli 1970)except that the separating gel contained 50 &ml fibrinogen and 2.0 A,, units/ml calf thymus DNA activated with DNase 62 bpI (Blank et al. 1983). After electrophoresis, the proteins were renatured using aqueous isopropanol, and catalytic activity was visualized by incubation of gels in the presence of a full complement of deoxynucleotides and “P-dTTP and autoradiography (Blank et al. 1983). For immunoprecipitation, synchronized cells were pulse labeled with either 0.1 mCi/ml [35S]methionine / or 0.1 mCi/ml [3ZP]orthophosphate at 37°C for 2 hr in methionine-free or phosphate-free culture media, reFIG. 1. Detection of P. falciparum DNA polymerspectively. Cells washed with cold phosphate-buffered ase activity on PCR and activity gel assay. (A) Top saline containing 10 mM methionine or phosphate lane: Reaction mixture (total 100 ul volume) of 0.1 were incubated in 10 mit4 Tris-HCI, pH 7.9, I mM pmole pBR 322, 10 pmole each of BamHI primers, 100 EDTA, and 5 mM DTT on ice for 20 min and centrifuged at 25,OOOgfor 20 min. The resulting pellet was nmole of dATP, dGTP, and dCTP, 10 uCi[a-32P]dTTP in 30 mMTris-acetate, pH 7.9,60 rruV sodium acetate, shown by DAPI staining to consist of intact nuclei. 10 mM magnesium acetate, and 10 mM DTT were inThis pellet was solubilized in 10mM Tris-HCI, pH 7.4, cubated at 100°C for 1 min and cooled to room tem1 mM EDTA, 2% Triton X-100,1 m&f PMSF, 10 t&ml leupeptin, and 10 t&ml pepstain A on ice for 30 min perature. Nuclear extracts (10 ~1) were added to the mixture and incubated for 5 min at room temperature. and centrifuged at 70,OOOgfor 1 hr. The supematant was preabsorbed with normal mouse serum (1: 1000 The PCR was repeated 10 times and after each incubation, lO-pl ahquots were removed for analysis on a dilution) in 10 vol of 20 mM Tris-HCl, pH 8.0,O. 13 M NaCl, 1 m&f EDTA, 0.5% Triton X-100, and 0.1 mg/ml 20% polyacrylamide gel. Size of DNA was determined BSA. Preabsorbed lysates were precleared with anti- by using pBR 322 Hue111digest (Sigma Chemical Co., St. Louis, MO). Bottom lane: Uninfected control cells mouse IgG-coupled Sepharose 4B, further incubated were treated in the same manner. (B) P. falciparum with anti-KB cell DNA polymerase-u antibodies nuclear extracts (lane 1) or control cell extracts (lane (1: 100 dilution), and immune complexes were precip2) were fractionated by SDS-polyacrylamide gel elecitated with the anti-mouse IgG-coupled Sepharose 4B. trophoresis (10% a&amide). Renaturation and detecAfter five washes with the immune precipitation tion of catalytic activity were done as described by buffer, followed by an additional wash with immune precipitation buffer containing 0.5 M LiCl, beads were Blank et al. (1983).

96

CHOI AND MIKKELSEN

TABLE I readily with themselves than with primers Effects of Aphidicolin on DNA Polymerase-a because of the relatively low molar ratio of Activity, DNA Synthesis, and Parasite Growth primers compared with target DNA (100: 1). DNA polymerase in P. falciparumConcentration % Inhibition” Measurement (eh4 infected cells was also detected on an activity gel (Fig. IB, lane 1). Two major bands DNA synthesis 23.5 0.1 (105 and 72 kDa) and two minor bands (180 1.0 68.4 5.0 86.1 and 130 kDa) exhibited catalytic activity. 0.1 30.6 As expected, no catalytic activity was ob- Polymerase activity 1.0 79.7 served when noninfected cells were used 82.9 5.0 (Figure lB, lane 2). No activity was ob- Growthb 0.01 16.3 served at approximately 40 kDa which by 0.1 93.6 1.0 100.0 size corresponds to a typical eukaryotic DNA polymerase+. DNA polymerase$ is Note. DNA synthesis and DNA polymerase activity also not found in some other protozoans were measured in the presence of various concentra(Fry and Leob 1986). tions of aphidicolin as described under Materials and Aphidicolin is a specific inhibitor for eu- Methods. To test the effects on Plasmodium growth, karyotic DNA polymerase-a, being ineffec- asynchronized cells were incubated in the presence of different concentrations of aphidicolin and parasitemia tive with the other DNA polymerases. We were determined by Giemsa staining. Culture medium tested the effects of aphidicolin on P. falcontaining aphidicolin was changed every 24 hr. Perciparum DNA synthesis, DNA polymerase centage inhibition represents the average of two sepactivity, and parasite growth (Table I). P. arate determinations. 0 Controls contained 0.1% Me,SO, the carrier for falciparum DNA synthesis and polymerase aphidicolin, and which caused no apparent growth inactivity were inhibited 60 and 79%, respec- hibition. tively, at a concentration of 1.0 I*g/ml b Initial parasitemia were 0.6% and final parasitemia aphidicolin. A slightly higher IC,, value for were calculated after 96 hr. Parasitemia were calcuaphidicolin inhibition of Plasmodium DNA lated based on counting 2000 cells. synthesis compared with those of other eukaryotes has also been previously reported teriophage, and animal viruses (Wong et al. (Janse er al. 1986). At this concentration of 1988). The sensitivity to aphidicolin sugaphidicolin, P. falciparum growth is com- gests that the Plasmodium DNA polymerpletely inhibited. This result supports the ase might react with antibodies against notion that P. falciparum may have a eu- mammalian DNA polymerase-cx. We have karyotic DNA polymerase-a. Since poly- used anti-KB cell DNA polymerase-a merase activity was not completely inhib- monoclonal antibodies which recognize ited by aphidicolin, the Plasmodium poly- epitopes on the 180-kDa catalytic subunit merase may be different from mammalian (as well as its degradation products) and DNA polymerase-cx in terms of sensitivity neutralize polymerase-a activity, but which to aphidicolin or, more likely, Plasmodium show no cross-reactivity with the p or 6 may have more than one kind of DNA poly- polymerases (Thommes et al. 1986; Wong merase, some of which are insensitive to et al. 1986; Tanaka et al. 1982). The results aphidicolin. However, the DNA polymer- of an immunoprecipitation experiment usase-o must be critical for replication and ing these monoclonal antibodies and metaasexual development. bolically labeled P. falciparum are shown A number of studies have shown that the in Fig. 2A (lane 1). Control experiments primary structure of DNA polymerase-a is with metabolically labeled cultures of nonhighly conserved between phylogenetically infected erythrocytes revealed no immunodistant species such as human, yeast, bac- precipitation with pooled antibodies. In

P. fdCipaW?l:

DNA

97

POLYMERASE-a

d g

1200-

g

lOOO-

VI z

600 -

c"

600 -

E 2

400 200 ;.--/-.

2

0 12

3

4

5

Pf stage FIG. 2. Immunoprecipitation with anti-KB cell DNA polymerase antibodies, activity gel, and DNA synthesis at different stages of P. falciparum development. (A) DNA polymerase-a was immunoprecipitated with pooled anti-human KB cell DNA polymerase-a monoclonal antibodies as described under Materials and Methods. Lane 1, anti-human KB cell DNA polymerase-a monoclonal antibodies (1:l mixture of SJK 132-20and SJK 287-38). Lane 2, nonimmune culture supernatant. Lane 3, SJK 132-20alone. Lane 4, nonimmune culture supematant. (B) Synchronized cultures (6 x lo6 cells/stage) were pulse labeled with [‘-%]methionine for 2 hr at 37°C and immunoprecipitated with pooled anti-KB cell DNA polymerase antibodies (SJK-132-20 and SJK-287-38) as described under Materials and Methods. The same cell preparations were used for both activity gel and DNA synthesis. (a) Total protein by Coomassie staining, (b) immunoprecipitation, (c) activity gel assay, and (d) DNA synthesis. Molecular weight markers are expressed in kilodaltons. Plasmodium stages: Stage 1, O-7 hr; stage 2, 7-12 hr; stage 3, 12-23 hr; stage 4, 23-27 hr; stage 5, 27-32 hr.

contrast with infected cultures, polypep- tion was also performed with the individual tides of M, 180, 165,105,72,52, and 45 kDa monoclonal antibodies. The results in lane were selectively immunoprecipitated and 3 of Fig. 2A show that SJK 132-20 by itself correspond in size to that observed with the immunoprecipitated Plasmodium DNA DNA polymerase-a of other eukaryotic or- polymerase-cy. As observed by Abuganisms. Among these, the 180-, 72-, and Elhiega et al. (1990), SJK 287-38 was inca52-kDa proteins appeared to react most pable of immunoprecipitating the Plasmostrongly with the antibodies. The 165- and dium enzyme (data not shown). The pooled antibodies also neutralized P. 105-kDa polypeptides probably represent degradation products of the 180~kDa sub- falciparum DNA polymerase activity maximally by 80% at an antibody concentration unit (Wong et al. 1986). Immunoprecipita-

98

CHOI AND MIKKELSEN

of 1.O kg/ml (Table II), similar to the degree of maximal inhibition obtained with aphidicolin. The residual activity remaining after treatment with antibody or aphidicolin suggests that Plasmodium has DNA polymerase activities other than the (Y form. We cannot rule out the possibility of coincidental differential sensitivities of human and Plasmodium DNA polymerases-ol to aphidicolin and antibody neutralization. The monoclonal antibodies were used to monitor the biosynthesis and post-translational phosphorylation of Plasmodium DNA polymerase-a during parasite maturation (Fig. 2B, lane b). Identical results were obtained whether metabolic labeling was performed at specific times after sorbitol lysis or if parasites at different stages of maturation were obtained by centrifugation on Percoll gradients. Plasmodium DNA polymerase-a was synthesized mostly at an early schizont stage (stage 4, 23-27 hr) as indicated by the appearance in the immunoprecipitates of [35S]methionine-labeled components at 180 kDa. There is some labeling in stages 3 and 5. Since the fractionation procedure for obtaining parasites at different stages of maturation is not absolute, we cannot be certain that this does not represent minor cross contamination of TABLE II Inhibition of Plasmodium DNA Polymerase Activity by Anti-human DNA Polymerase-a Antibodies Antibody (wdml) 0.0 (control) 0.01 0.1 1.0 10.0

Radioactivity (cpm) Expt 1

Expt 2

% Inhibition

1748 654 438 349 410

2739 999 764 574 621

0.0 63.0 74.4 81.7 76.9

Note. Nuclear extracts were incubated in the presence of the different concentrations of pooled antihuman DNA polymerase antibodies at 4°C for 30 min. After precipitation of the immune complex with antimouse IgG-coupled Sepharose 4B, the resulting supernatants were assayed for DNA polymerase activity as described under Materials and Methods.

these fractions by stage 4 parasites. At stage 4, the catalytic activity of the DNA polymerase also appears and maximal activity is maintained through stage 5 (Fig. 2B, lane c). The appearance of newly synthesized polymerase-ti and activity at stage 4 corresponds with the commencement of DNA synthesis (Fig. 2B, lane d). We could not detect any phosphorylation of the immunoprecipitated polymerase subunits except the 52-kDa protein and this appeared concomitantly with its synthesis (data not shown). These data indicate that P. falciparum synthesize DNA polymerase-ti only at a specific time in the early schizont stage of maturation and that this DNA polymerase is necessary for chromosomal replication. In summary, our results demonstrate that P. falciparum synthesize a typical eukaryote DNA polymerase-cx in terms of aphidicolin sensitivity in agreement with previous studies (Inselburg and Banyal 1984; Janse et al. 1986; Abu-Elhiega et al. 1990) and subunit structure. Abu-Elhiega et al. (1990), using one of the monoclonal antibodies against the KB cell DNA polymerase-a employed here (SJK 287-38), were unable to neutralize Plasmodium polymerase-cy activity, but by use of activity gels and partial purification by molecular sieve chromatography demonstrated a DNA polymerase activity with an approximate M, of 100 kDa. The present analysis using immunoprecipitation with monoclonal antibodies against human DNA polymerase-a and activity gels suggests that Plasmodium polymerase in common with that of other eukaryotic cells is composed of a large 180-kDa catalytic subunit and that the lOO-kDa component found by AbuElhiega et al. (1990) is probably a degradation product. We were unable to demonstrate phosphorylation of the 180-kDa catalytic subunit, suggesting that phosphorylation of this subunit is not a key factor in the regulation of DNA polymerase activity as ap-

P. fakiparldm:

DNA

pears to be the case with some other cells. The close temporal relationship between the synthesis of the MO-kDa component, the appearance of catalytic activity, and DNA synthesis would suggest that in P. falCiparum regulation of DNA polymerase activity is primarily at the transcriptional level. At present we have no evidence indicating that the activity of the Plasmodium enzyme like other DNA polymerases-ol is modulated by association with accessory or cofactor proteins (e.g., Pritchard et al. 1983; Got&an and Heard 1990). A specific antibody against the catalytic subunit of Plasmodium DNA polymerase as demonstrated here provides a tool for investigating these associations as well as the transcriptional regulation of activity. Finally, we note that only one of two monoclonal antibodies against human DNA polymerase-a immunoprecipitates the Plasmodium enzyme, suggesting that there are structural differences between the host and parasite enzymes. These may be potentially exploitable in terms of developing novel antimalarial chemotherapy. ACKNOWLEDGMENT

Research was supported by Grant AI24307 from the United States Public Health Service. REFERENCES

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LAEMMLI, U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685.

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MULLIS, K. B., AND FALOONA, F. A. 1989. Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction. In “Recombinant DNA Methodology” (R. Wu, L. Grossman, and K. Moldave, Eds.), pp. 189-204. Academic Press, New York. PRITCHARD, C. G., WEAVER, D. T., BARIL, E. F., AND DEPAMPHILIS, M. L. 1983. DNA polymerase alpha cofactors C’C2 function as primer recognition proteins. Journal ofBiological Chemistry 258,98109819.

READ, L., AND MIKKELSEN, R. B. 1990. Cyclic AMPand Ca2+-dependent protein kinases in Plasmodium falciparum. Experimental Parasitology 71, 3948. TANAKA, S., Hu, S., WANG, T. S., AND KORN, D. 1982. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. Journal of Biological Chemistry 257, 83864390.

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MIKKELSEN

Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells. Biochemistry 25, 1308-1314. WONG, S. W., PABORSKY, L. R., FISHER, P. A., WANG, T. S., AND KORN, D. 1986. Structural and enzymological characterization of immunoafftnitypurified DNA polymerase alpha-DNA primase complex from KB cells. Journal of Biological Chemistry 261, 79587968. WONG, S. W., WAHL, A., YUAN, P., ARAI, N., PEARSON, B. E., ARAI, K., KORN, D., HUNKAPILLER, M. W., AND WANG, T. S. F. 1988. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic DNA polymerases. EMBO Journal

7, 3747.

Received 10 October 1990; accepted with revision 27 March 1991