Eur. J. Biochem. 93, 271 -278 (1979)

DNA Polymerases of Anucleated Cells Isolation and Characterization of Two DNA Polymerases from Human Platelets Laura TARRAGO-LITVAK, Claude DESGRANGES, Alejandro ARAYA, and Simon LITVAK Dtpartment de Biochimie, Universitt de Bordeaux 11, et Unite no. 8 de l’lnstitut National de la Santt et de la Recherche Medicale (Cardiologie), Pessac (Received July 31 /October 24, 1978)

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase y type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called ‘cytoplasmic’ DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase M from normal cells : DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimideand the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase 8. DNA synthesis takes place in two well-defined compartments in a normal animal cell: the nucleus and the mitochondria [l]. Three DNA polymerases, M , p and y, have been purified and characterized from whole animal cells. The most recent reviews on eukaryotic DNA polymerases are [2-51. The study of the subcellular distribution of DNA polymerases a and j? indicates that when the extraction process is done under usual aqueous extraction conditions, DNA polymerase a is found in the soluble cytoplasm and in’ the nucleus; DNA polymerase p is strongly bound to chromatin, and thus it is found in the nuclear fraction, while DNA polymerase y is found in the nuclear, soluble cytoplasmic and mitochondrial fractions. In the mitochondria, DNA polymerase y seems to be the predominant, if not unique, activity [6 - 81. The presence of DNA polymerases M and p in the cytoplasm is thought to be an artifact arising from the damage of nuclear envelopes during homogenization. It has been reported that when techniques like non-aqueous cellular extraction (100 % glycerol), or nuclei extrusion by cytochalasin B are used, most of the DNA polymerase M and p activities are found associated to the nuclear fraction [9 - 101. Nuclei are far from pure under these conditions and some cytoplasmic contamination may be present. If we assume that DNA Abbreviation. I-b-D-Arabinofuranosyl cytosine 5’-triphosphate, aCTP. Enzymes. DNA polymerase (EC 2.7.7.7); pancreatic DNase (EC 3.1.4.5).

polymerases are synthesized in the cytoplasm, their presence in this compartment is not surprising. The level of DNA polymerase M and y (nuclear) are closely related to the cell cycle. These two activities are increased during the S phase of active DNA synthesis, while the level of DNA polymerase p does not change during the cell cycle [11- 131. The search for a suitable system to study the nuclear control of mitochondrial DNA polymerase activity led us to the utilization of human platelets. These anucleated cells are produced by cytoplasmic division of megakaryocytes [14,15]. Protein synthesis, as well as nucleic acid synthesis, takes place in these cells [16,17]. In this article we describe the isolation, purification and characterization of two DNA polymerases from human platelets. One of them is found mainly in the mitochondrial fraction, although some activity is also found in the extramitochondrial cytoplasmic fraction. All the properties of this enzyme are identical to those of DNA polymerase y from normal cells. The other activity is found only in the extramitochondrial cytoplasmic fraction and cannot be classified as a typical M or p DNA polymerase. A preliminary report of this study has appeared [ 181. MATERIALS AND METHODS Mu t erials Radioactive precursors of nucleic acids were obtained from the Radiochemical Centre (Amersham).

Human Platelet DNA Poiymerases

272

Nucleosides, N-ethylmaleimide, ethidium bromide and 1-/?-D-arabinofuranosyl cytosine 5'-triphosphate (aCTP) were purchased from Sigma Chem. Co. Phosphonoacetic acid was a kind gift from Drs Chousterman and Girard. DEAE-cellulose and phosphocellulose were from Whatman. Nitrocellulose filters (0.45 pm) were from Schleicher and Schull. Calf thymus DNA, poly(A), poly(dA) and DNase I were from Sigma. Poly(dC), oligo(dT)lZ and oligo(dG)12 were from Boehringer Mannheim.

Purification of Human Platelets

Three platelet concentrate containers (about 1 1 of blood) were centrifuged for 15 min at 150 x g to remove erythrocytes and nucleated cells. The supernatant platelet-rich plasma was centrifuged at 1100 x g and platelets were resuspended in a buffer solution containing 1 volume of 250 mM Tris-HC1 pH 7.4 and 2 volumes of 150 mM NaC1, 25 mM EDTA. Platelets were resedimented by centrifugation and resuspended in the same buffer. This operation was repeated three times.

Purification of Mitochondria

a ) Sonication Method. Cells were resuspended in 250mM sucrose, 10mM Tris-HC1 pH 7.4, 2 m M EDTA. The suspension was sonicated at 4 "C for 10 s, several times, in an Annemase Ultrasonic disintegrator at maximal intensity. The extent of breakage was followed in a phase-contrast microscope. Cell lysates were centrifuged twice at 1OOOxg and the supernatant was centrifuged for 20 rnin at 10000 x g. The pellet corresponded to an enriched mitochondrial fraction. b ) Hypotonic Lysis Method. The method described by Barber and Jamieson was used [19]. It consists essentially of centrifuging platelets through a glycerol gradient (0 - 4.3 M) under controlled conditions; this process results in the internal equilibration of the platelets with the glycerol. The glycerol-filled cells are then submitted to hypotonic lysis in order to release the intracellular material. Preparation of Subcellular Fractions

The disrupted cells obtained by hypotonic lysis were centrifuged 10 min at 1000 xg. The pellet was resuspended in 10mM Tris-HCl pH 7.5, 250mM sucrose (1000 x g fraction) and the supernatant was centrifuged at 10000 x g for 10 min. This pellet was resuspended in the same buffer (mitochondrial fraction), while the supernatant was kept as the cytoplasmic fraction. All fractions were sonicated in the

presence of 0.05 % Triton X-100 before measuring the enzymatic activity. Electron Microscopy

Platelets and subcellular fraction were fixed in 2.5 % glutaraldehyde in 100 mM potassium phosphate pH 7.4 for 45 rnin in the cold, rinsed with buffer, post-fixed in 1 % OsOe for 60 min at 4 "C, dehydrated in ethanol and propylene oxide and embedded in Epon 812. Sections obtained with a Reichert ultramicrotome were stained with uranyl acetate followed by lead citrate and observed with a Philips SM 201 electron microscope. Partial Purijkation of Human Platelet DNA Polymerases

Total extract was prepared by soncication of 2 x 10l1 pelleted freshly prepared platelets in 1 niM potassium phosphate pH 7.5, 1 mM 2-mercaptoethanol, 0.2% Nonidet P-40 and 20% glycerol. The suspension was sonicated three times for 10 s at 4°C. The extent of the breakage was followed by phasecontrast microscopy. After centrifuging for 15 min at 10 000 x g, the precipitate was again sonicated and all the soluble material was adsorbed on a DEAEcellulose column equilibrated with 20 mM potassium phosphate pH 7.5, 1 mM 2-mercaptoethanol, 0.2 % Nonidet P-40 and 20 % glycerol (buffer A), washed extensively with the same buffer and eluted with a linear gradient from 20 to 500mM phosphate in buffer A. The fractions having enzyme activity were pooled together, dialyzed and adsorbed on a phosphocellulose column equilibrated with buffer A. After washing the column, elution was performed with a linear gradient from 20 to 500 mM phosphate. DNA Polymerase Assay In a final volume of 50 p1 the incubation mixture contained 50 mM Tris-HC1 pH 8.0, 8 mM MgClz or 0.5 mM MnC12, 30 pg/ml activated DNA, 0.05 mM each of dATP, dCTP and dGTP, 0.5- 10 pM [methyl3H]thymidine 5'-triphosphate (specific activity 200 2000 counts min-' pmol-l) and 10 pg/ml bovine serum albumin. When poly(A) . (dT)12 was used as template, the reaction was done in the presence of 50mM potassium phosphate, manganese ions and 20 pg/ml of polynucleotide [7]. Incubation was carried out at 37°C for different lengths of time. Reactions were stopped by addition of 0.1 ml of ice-cold 20% trichloroacetic acid containing 1 % sodium pyrophosphate. The precipitates were filtered on nitrocellulose membranes, washed with 5 % cold trichloroacetic acid, dried and counted in a PPO/POPOP/toluene scintillation mixture.

273

L. Tarrago-Litvak, C. Desgranges, A . Araya, and S. Litvak

D N A Activation

Calf thymus DNA was activated by treatment with pancreatic DNase I as described by Aposhian and Kornberg [20]. Preparation of Synthetic Double-Stranded Polynucleotides

Double-stranded synthetic polynucleotides used as template were prepared from single-stranded material as described previously [21]. Molecular Weight and Sedimentation Coeflicient Determination

The sedimentation coefficient was determined by sucrose gradient centrifugation [22] and the molecular weight by gel chromatography [23] in a Sephadex G-150 column. Enzyme Neutralization Assay

Antibody against DNA polymerase CI from HeLa cells was a kind gift of Dr A. Weissbach. The assay was performed as described previously [27]. RESULTS SubceIIuIar Fractionation of Human Platelets

Two different methods have been used to isolate platelet organelles : osmotic shock of glycerol-filled platelets or sonication of intact platelets. Fig. 1A shows our starting material; no contamination with nucleated cells could be observed by phasecontrast microscopy, while erythrocytes represented less than 0.1 % of the platelet population. In Fig. 3 B, where an enlarged platelet is observed, we can clearly distinguish the absence of nuclei and the presence of a few mitochondria per cell, as well as dense granules. After sonication and centrifugation, as described under Methods, we obtained a mitochondria-enriched pellet (Fig. 1C) with membrane vesicles and some granules still present. Mitochondria used for the purification of DNA polymerase were structurally well preserved as seen in Fig. 1 D. Subcellular Distribution of Platelet D N A Polymerase Activity

By using the glycerol centrifugation method, we were able to isolate three subcellular fractions the 1000 x g pellet, the mitochondrial pellet or 10000 x g and the postmitochondrial supernatant or cytoplasmic fraction. Two DNA polymerase assays were performed, one using activated DNA as template and the otherming poly(A) . (dT)lz. Table 1 shows that the main activity of the cytoplasmic fraction is found with activated

DNA. When the 1000 x g or the mitochondrial fraction was assayed, a much higher activity was found with a poly(A) . (dT)lz template. Partial Purijicat ion of Platelet D N A Polymerase Activities

Chromatography on DEAE-cellulose was performed with a total platelet extract (Fig.2A) or with a mitochondrial extract as described under Methods (Fig. 2B). It can be seen that when the total extract was examined, two clearly distinct peaks of activity were found; one preferentially used poly(A) . (dT)lz and the other was more active in the presence of activated DNA. In the mitochondrial extract the only activity we detected was the one that recognizes preferentially a poly(A) . (dT)lz template (DNA polymerase y).When 1000 x g extract was submitted to the same type of chromatography, the pattern obtained was identical to the one in Fig.2B (not shown). Both activities have been further purified by phosphocellulose chromatography as described in Methods ; y polymerase was eluted at 0.3 M potassium phosphate and the ‘cytoplasmic’ enzyme at 0.25 M. A representative purification of ‘cytoplasmic’ DNA polymerase and y polymerase from human platelets is shown in Table 2. Properties of D N A Polymerases y and Cytoplasmic from Human Platelets

Both DNA polymerases have high molecular weight : about 120000 for the ‘cytoplasmic’ enzyme and 150000 for y, as determined by Sephadex chromatography (not shown). By sucrose gradient centrifugation a sedimentation coefficient of about 7 for DNA polymerases y and ‘cytoplasmic’ was obtained (Fig. 3). The sedimentation coefficient was determined in 500 mM KC1 in order to avoid artifactual associations. The template specificity of both enzymes is shown in Table 3. Activated DNA is used by the two polymerases more efficiently in the presence of Mg2’ than Mn2’ ; the same is observed with poly(dA) . (dT)lz and poly(dC) . (dG)12. As expected, poly(A) . (dT)lZ is an excellent template for DNA polymerase 7 . With poly(C) . (dG)lz, a very good template of oncornavirus DNA polymerases, no activity at all was detected with human platelet DNA polymerases. The same negative results were obtained when a poly(dT) . poly(A) (template-primer) was used and dAMP polymerization was followed. As a control we assayed mouse cells (EMT-6 cells) DNA polymerase CI and 6, [21]. DNA polymerase CI from mouse cells elongates very efficiently a synthetic RNA primer, while the enzyme is not active in this system ([24] and our unpublished results). As seen in Fig.4, the antibodies against HeLa DNA polymerase CI inhibit very efficiently DNA poly-

274

Human Platelet DNA Polymerases

Fig. 1. ( A / Elecfron micrograph of platelet-rich plasma ( x 1350O). See Methods. ( B ) Platelet mapiji’cation ( x 30000). Mitochondria are indicated by arrows. ( C ) Electron micrograph of’ the mitochondriu-enriched fiaction prepared as described in Methods (x3OOO0). (0)Mugnijkution from the mitochondria1fraction ( x 135000)

merase CI from EMT-6 cells and only partially the ‘cytoplasmic’ DNA polymerase from human platelets. When the activities of both D N A polymerases from platelets were tested at different concentrations of salt, a stimulation was obtained with an optimum of about 40 mM KCl in both cases. Strong inhibition is observed at higher concentrations and little activity is detected at 200 mM (not shown). The ‘cytoplasmic’ DNA polymerase shows a remarkable stability at 45 “C as shown in Fig. 5. After 30 min of incubation at 45 “C, DNA polymerase y

has lost more than 80 % of its original activity, while practically no change is observed with the ‘cytoplasmic’ enzyme. The behaviour of D N A polymerases y and ‘cytoplasmic’ from human platelets in the presence of different inhibitors of DNA polymerases is shown in Fig. 6. The inhibition of DNA polymerases CI and p from mouse cells [21] is shown in the same figure. We have not included the inhibition of DNA polymerases y from mouse cells, since the pattern in all cases is identical to the y polymerase from platelets. It can

L. Tarrago-Litvak, C. Desgranges, A. Araya, and S. Litvak

275

be seen that the ‘cytoplasmic’ polymerase from human platelets is extremely resistant to aCTP and N-ethylmaleimide, moderately affected by ethidium bromide and very sensitive to phosphonoacetic acid.

I

I

-z. OI

c

0.5 ,“

a

0

L Q

DISCUSSION The purpose of this study was to isolate and characterize the DNA polymerases from human platelets. These blood cells are produced by cytoplasmic division of megakaryocytes in the bone marrow. They are anucleated, but contain mitochondria and different kinds of granules. It seemed to us that platelets could be a good model system for the study of mitochondrial DNA synthesis in the absence of nuclear control. We were encouraged by the fact that DNA polymerase y from mitochondria of normal cells was much more stable than DNA polymerase y from the nuclear fraction of the same cells, as judged by the effect of cycloheximide, an inhibitor of cytoplasmic protein synthesis (our unpublished results). We started, then, the search for a mitochondrial DNA polymerase in human platelets. To our surprise we found, in platelets, a second activity clearly different from the mitochondrial enzyme.

E, - 0.3 ‘2 m

c

n“ -

0

20

0.1

80

40 60 Fraction number

Table 1. Cellular distribution of platelet D N A polymerase activity Subcellular fractions and assays were done as described in the text. Incubation time was 60 min. Specific activity was measured as the amount of dTMP incorporated/mg protein Fraction

Poly(A) . (dT)12

Activated DNA

.-

Cytoplasm 1000 x g pellet 10000 x g pellet

~-

~

percentage of total

spec. act.

pmol/mg

X

pmol/mg ”/,

46 140 284

53 18 29

spec. act.

.

35 395 890

percentage of total

21 32 41

0

10

20

30

40

50

60

Fraction number

Fig. 2. Chromatographic prqfile on a DEAL-cellulose column (4’ human platelet D N A polymeruse activities. (A) Total extract, or (B) mitochondrial fraction prepared as described under Methods was loaded separately on to a DEAE-cellulose column (2 x 9 cm). Elution and DNA polymerase assay were carried out as described in the text. After extensive washing, a linear gradient of ten times the bed volume of the column was used. (0-0) Activated DNA; (A----A) poly(A) (dT)12

Table 2. Partial purification of platelet DNA polymerases 1 unit of enzyme incorporated 1 nmol 13H]dGMP/h using poly(dC). ( d Q l 2 . Assays were done as described under Methods, using 20 &ml poly(dC) . (dG)12and MgA2as cation. The specific activity of [3H]dGTP was 250 counts min-I pmol-’ Enzyme

Fraction

Volume

Protein

Specific activity

Purification

ml

mg/ml

units/mg

-fold 1 10

‘Cytoplasmic’ DNA polymerase

crude extract DEAE-cellulose (pooled peak) phosphocellulose (pooled peak)

58 56 13

3.3 1.2 0.7

0.06 0.62 4.21

70

DNA polymerase y

crude extract DEAE-cellulose (pooled peak) phosphocellulose (pooled peak)

58 36 23

3.3 1.2 0.1

0.05 0.46 5.25

1 9 105

Human Platelet DNA Polymerases 60

c

0 .r

"

. .P

+

r Y)

c

40 2 c1

b 8 m 'A m L

0

20

gE, 4

Z

0

aJ Y) 3

K

0 0

ao

40

120

A n t i - a (pg)

Fig. 4. Neutralization effect of HeLa cells D N A polymerase c( antibody on human platelet and mouse cells D N A polymerase a:Assays were performed as described under Methods. Percentage residual activity of: (A-A) mouse cells (EMT-6 cells) DNA polymerase cc; (A------A) human platelets 'cytoplasmic' DNA polymerase

0 0

5

10

15

20

25

30

35

I

F r a c t i o n number

Fig 3. Sedimentation velocity centrijugation. 4-ml linear sucrose gradients were made with 5 - 20 "/, sucrose (w/v) in 50 mM TrisHCI pH 7.5, 1 mM EDTA, 2 mM 2-mercaptoethanol and 500 mM KCI. Human immunoglobulin (7.2 S), cytochrome c (2 S) and tRNA (4 S) were used as markers. Centrifugation was carried out in a MSE ultracentrifuge, for 15 h at 53000 rev./min in a 6 x 5 rotor and the temperature was held at 0-4°C. Fractions were collected by peristaltic pump aspiration from the top of the gradients. 10-p1 aliquots of each fraction were assayed using poly(dA). (dT)12 as template. (-0) 'Cytoplasmic' DNA polymerase; (+----+) mouse cells (EMT-6) DNA polymerase a ; (&-----A) mouse cells (EMT-6) DNA polymerase Table 3. Template Specificity of Human Platelet D N A Polymerases Assays were done as described under Methods, using 20 pg/ml of each polynucleotide and 170 pg/ml of activated DNA. Incubations were carried out for 60 min Template

'Cytoplasmic' DNA polymerase

DNA polymerase y

MnZi

Mn2+

Mgz+

-_ _ ~ Mgz+

pmol incorporated Poly(dA). (dT)iz (5: 1) Poly(A) . (dT)lz (5: 1) Poly(dC) . (dG)iz (5: 1) Activated DNA Poly(C) . (dG)iz (5: Poly(dT).poly(A)(lO:l)b

8.6 3.5 28.9 4.4 < 0.1

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Eur. J. Biochem. 93, 271 -278 (1979) DNA Polymerases of Anucleated Cells Isolation and Characterization of Two DNA Polymerases from Human Platelets L...
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