Stimulation of purified DNA polymerase α by various basic proteins which interact with activated DNA

ANALYTICAL

BIOCHEMISTRY

166,361-367

(1987)

Stimulation of Purified DNA Polymerase CYby Various Basic Proteins Which Interact with Activated DNA’ :rAKASHl

HIRONAKA,* TAKEYOSHI

ASAKO

ITAYA,

MINAGA,*

Received

AND

February

KOICHIRO TOMOYA

YOSHIHARA, KAMIYA

18. 1987

Extensive purification of DNA polymerase n-primase resulted in a marked loss of the DNA polymerase LYactivity. This loss is due partly to the elimination of some basic proteins from the enzyme preparation since the activity of purified enzyme was stimulated lo- to I S-fold by the addition of various basic proteins, including all five classes of histones. protamine, poly-L-lysine. and poly-L-arginine. at a concentration of 2 fig/O.? ml in the presence of 20 &0.2 ml of activated DNA. The optimum concentration of the basic proteins and the maximum activity attained at that concentration varied with varying concentrations of the template primer used. indicating that the observed stimulation is caused by an interaction between these basic proteins and activated DNA. The enzyme activity with an optimal concentration of activated DNA was markedly inhibited by the addition of denatured DNA. The suppressed enzyme activity could be restored by an appropriate concentration of histone H 1. These results suggest that histone H I and other basic proteins protect the enzyme from forming an abortive complex with singlestranded DNA or with a long stretch of the single-stranded part of activated DNA as singlcstranded DNA-specific binding proteins do (M. Sapp, H. Kiinig, H. D. Riedel. A. Richter, and R. Knippers ( 1985) J. Biol. C!iem. 260, 1550-1556). Spermine also showed a similar stimulatory e&t. All acidic proteins tested were ineffective. :C 1987 Academic Press. Inc. KEY WORDS: DNA polymerase n: stimulation: histones: protamine: homopolyamino acids: spermine.

Accumulating evidence indicates that a species of DNA polymerase N carrying a tightly bound primase is widely distributed among various eukaryotic cells ( l-6). In an attempt to purify the DNA pol a-primase’ from bovine thymus, we found that the recovery of the (enzyme activity was extremely low, especially at an advanced step of purification. in spite of our effort to protect the enzyme from proteolytic attack. Since several reports (7-12) have indicated the pres’ This work was supported in part by Grant-in-Aid for Scientific Resea,rch 6 1480 I34 and Grant-in-Aid for Cancer Research 6 IO 10089 from the Ministry of Education, Science. and Culture of Japan. * Abbreviations used: DNA pol a-prim=. DNA polymerase o( with primase activity; sDNA. single-stranded DNA.

ence of some specific stimulating factors of DNA polymerase LYin various cells. we attempted to find such a factor, which may be lost from the purified enzyme. Thus. we tested several side fractions obtained at various purification steps for their DNA pol (Ystimulating activity. Surprisingly. many of these fractions were more or less active in stimulating the enzyme activity, suggesting that a wide variety of proteins may have similar effects. Therefore, we examined various known proteins for DNA pol cu-stimulating activity and found that all basic proteins tested, including histones, protamines. poly-L-lysine, and poly-Larginine, were almost equally effective and stimulated the enzyme activity IO- to 15fold, while all nonbasic proteins tested were

362

HIRONAK4

ineffective. Since our assay system for DNA pol cycontained a relatively high concentration (0.2 mg/ml) of bovine serum albumin to protect the enzyme from physical denaturation, the observed effect may be due to a mechanism other than enzyme stabilization. Thus, the mode of stimulation of DNA pol N by these basic proteins was studied using mainly histone HI as protein. MATERIALS

AND

METHODS

Materials [r7?ethy/-3H]dTTP and [S(N)-3H]dATP were obtained from New England Nuclear. Histones were prepared by the method of Bijhm et al. (13). Homopolyamino acids were products of Sigma. Casein, y-globulin. ovalbumin, protamine sulfate, and spermine were obtained from Nakarai Chemicals, Ltd. (Kyoto, Japan). Activated calf thymus DNA was prepared according to the method of Mayer and Simpson (14). Poly(dT) was obtained from Sigma. Esckichia coli DNA polymerase I was purchased from Seikagaku Kogyo, Co.. Ltd. (Tokyo. Japan). Bovine thymus DNA polymerase 6 was purified according to the method of Chang (15). DNA pot cu-primase was purified from crude extracts of bovine thymus by a conventional method including phosphocellulose. Q-sepharose. sDNA-cellulose, and Sephacryl S-300 column chromatographies. Approximately a IOOO-fold purification was achieved with a recovery of 15%. The specific activity of the enzyme was 8000 units/mg. The DNA polymerase activity was sensitive to aphidicolin and N-ethylmaleimide and resistant to ddTTP. The enzyme was associated with primase and showed a high DNA replicase activity in the presence of a specific primasestimulating factor.3 ‘A. Itaya, T. Hironaka. and K. Yoshihara. manuscript in preparation.

El 41.

. l.s.str~~f. The assay of DNA pol (r activity with acti\,ated DNA as a template primer was carried out essentially as described by Matsukage ( 16). The assay mixture contained 50 mM Tris-HCI buffer, pH 7.4: 6 mM MgCl,; I mM dithiothreitol; 1 mM ATP: 20 ,UM dATP. dGTP, and dCTP: 20 PM [mc?h>$--?H]dTTP (32 cpm/pmol); 16% (v/v) glycerol: 40 pg of bovine serum albumin; 50 mM KCl: 20 PLg of activated calf thymus DNA; and an appropriate amount of the enzyme in a total volume of 0.2 ml. In some experiments, the concentation of activated DNA was varied and various basic proteins were added as indicated in the text. The reaction was carried out at 37°C for 30 min. Acid-insoluble radioactivity was determined as previously described (17). One unit of enzyme activity is defined as 1 nmol of deoxyribonucleotide incorporated into acid-insoluble products during a 60-min incubation of the mixture supplemented with 4 hg of histone H 1. The assays of DNA polymerase 8 and E. co/i DNA polymerase I also were carried out as above, except that the mixture for DNA polymerase p contained SO mM TrisHCI buffer, pH 8.0, in place of the buffer of pH 7.4. A.s.sa)-II. DNA replicase activity was assayed with poly(dT) as template and without exogenous primer principally as described by Yagura ef ~11.(3). The standard reaction mixture contained 50 mM Tris-HCl buffer. pH 7.4, 10 mM MgCI?. 1 mM dithiothreitol, 40 pg bovine serum albumin, 1 mM ATP, 0.02 ODU of poty(dT). 20 fiM [3H]dATP (33 cpm/pmol), 10 ng of primase-stimulating factor, and 2.5 units of DNA pal cr-primase in a total volume of 0.2 ml. The reaction was carried out at 37°C for 60 min. RESULTS

In an attempt to extensively purify DNA pol Lr-primase from bovine thymus, we found that the recovery of DNA polymerase activity in purified enzyme was extremely

STIMULATION

OF

DNA

POLYMERASE

low, suggesting that some stimulating factor was eliminated. Thus, we tested several side fractions eliminated from the enzyme during the purification for their enzyme-stimulating ability. Contrary to our expectation, many fractions, including a fraction eluted at 1 M NaCl from phosphocellulose column, a passthrough fraction from phenyl-Sepharose, and two protein peaks eluted from sDNAcellulose column, all showed stimulator-y activity. although their stimulating efficiencies were fairly different. Among the endogenous protein fractions tested, the phosphocellulose fraction eluted at 1 M NaCl possessed the highest stimulating efficiency, suggesting that the stimulating proteins may have basic natures. Thus, we examined various known basic proteins for their enzyme-stimulating activity. Surprisingly, all of the tested basic proteins, including five histones, protamine, and basic homopolyamino acids, poly-L-lysine and poly-L-arginine. showed an efficient

PROTEIN

'gg)

POLYAtlINO

ACID

bg)

FIG. 1. Effect of various proteins and homopolyamino acids on DNA polymerase 01 activity. (A) The indicated concentrations of histone HI (0). H2A (A). H2B (A). H3 (0) H4 (Cl), casein (m). y-globulin (0). ovalbumin (x), and an endogenous fraction eluted from phosphocellulose column at 1 M NaCl (---) were added to the reaction mixture containing 2.5 units of purified DNA polymerase cy. (B) Poly-L-lysine (0). poly-L-arginine (0). protamine (A), poly-t-glutamic acid (A). and poly-Laspartic acid (0) were tested as above.

CY BY BASIC

363

PROTEIN

0.1

I SPERMINE

Ii)

(ml?)

FIG. 2. Effect of spermine. The activity of 1.5 units of enzyme was assayed in the presence of the indicated concentration of spermine.

stimulation similar to that ofthe endogenous fraction eluted from the phosphocellulose column: the observed stimulation at 2 pg/O.2 ml of these proteins was lo- to 15-fold (Figs. IA and 1B). In addition to these basic proteins, another basic compound, spermine, was also effective: the maximum stimulation attained at 1 mM was near the level attained at optimum concentrations of histone Hl and other basic proteins (Fig. 2). Spermine, however, did not alter the optimum concentration of Mg’+ (data not shown). The addition of histone H 1 in the presence of 1 mM spermine did not appreciably stimulate the enzyme activity further. In contrast to these basic compounds, all of the nonbasic proteins tested including casein, y-globulin, ovalbumin, poly-L-glutamic acid, and polyL-aspartic acid were not effective at all (Fig. 1). These results indicate that the positive charge carried by the above basic polymers is a prerequisite for the observed stimulation. Thus, we further examined the mode of stimulation using histone Hl as a representative of various basic proteins. As shown in Fig. 3, the stimulator-y effect of histone H 1 on DNA polymerase CYactivity could be observed onlywhen a highly purified enzyme was used. As expected from findings that various protein

364

HIRONAKA

10 ENZYME (rg)

20

0.2

0.4

0.6

ENZYME (114)

FIG. 3. Effect of histone HI on the activity of a crude and a highly purified DNA polymerase N. The activities of (A) the crude enzyme at the phosphocellulose column chromatography step and of (B) the purified enzyme were assayed in the presence (0) and absence (0) of 4 pg/O.Z ml of histone HI in the reaction mixture.

fractions eliminated from the enzyme showed the enzyme-stimulating activity, the activity of the crude enzyme was not affected by the addition of histone HI to the assay system. The observed stimulation of DNA polymerase activity seems to be a phenomenon rather specific for DNA polymerase N species. since both of the activities of highly purified bovine thymus DNA polmerase B and E. coli DNA polymerase I were not affected by the addition of the concentration of histone HI (data not shown). In order to prove that the effect of basic proteins is not due to enzyme stabilization. we examined the effect of the addition of histone H 1 at various times after initiation of the enzyme reaction. As shown in Fig. 4, the addition of histone H 1 to the reaction mixture at any time after the start of incubation could immediately initiate a high and constant rate of DNA synthesis, indicating that the low activity observed in the absence of histone Hl was not the result of an irreversible inactivation of the enzyme molecule itself. An effect of histone H 1 as a carrier for acid precipitation of the product DNA also could be negated since its addition at the end of incubation was totally ineffective (Fig. 5).

ET Al

When histone Hl concentration was varied in the presence of a hxcd concentration of activated DNA (20 pg/O.Z ml). the maximum activity was attained at 4 pg/O.Z ml, and a further increase in histone conccntration resulted in a rapid loss of the activity (Fig. 5). When the concentration of activated DNA was decreased to 6.7 and 2.2 pg/O.2 ml, the optimum histone concentration also decreased to 1 and 0.3 pg/O.2 ml. respectively (Fig. 5). Thus, the optimum concentration of histone Hl paralleled the concentration of activated DNA in the reaction mixture. and maximum stimulation was observed at an approximately constant histone H 1/DNA ratio (w/w) of 0.3, indicating that the observed stimulation was caused by an interaction of the basic protein with the template primer DNA. Next. we examined the enzyme activity by varying the concentration of activated DNA in the presence of histone Hl at a constant histone H l/activated DNA ratio of 0.7 (w/w) and compared the DNA effect to that without histone H 1. As shown in Fig. 6A. at concentrations lower than 1 pg/O.2 ml, activated DNA stimulated the enzyme activity a!most

“: 0

0

30 INCUBATION

60

90

TIME cmin)

FIG. 4. Effect of histone Hl on the time course of DNA synthesis by purified DNA polymerase tv. Two units of purified enzyme was incubated as described under Materials and Methods, except that 4 rg/O.2 ml of histone HI was added to the reaction mixture 0 (0). 30 (a), and 60 min (A) after the start of the incubation or the reaction was carried out without histone HI (0).

STIMULATION

OF

DNA

POLYMERASE

equally regardless of the presence of histone H 1. In the absence of histone H 1, however. the enzyme activity reached a maximum at 2.5 wg/ml, and a further increase in the DNA concentration inhibited the enzyme activity. while in the presence of histone H 1, the activity continued to increase until the DNA concentration reached more than 20 pg/O.2 ml. A double reciprocal plot of the titration curve obtained without histone Hl clearly indicated that a high concentration of activated DNA functions as a strong inhibitor and. thus, the plot is nonlinear, while the plot obtained in the presence of histone HI and activated DNA at a constant ratio is linear (Fig. 6B). Based on the latter plot, the apparent K,,, for activated DNA and the I,,, were calculated to be 8.5 ccg/O.2 ml and 11,250 cpm/30 min/2 units of the enzyme, respectively. The I’,,,, value corresponds to approximately 1.4 times that attained in the

0

0.31

12 I

I HISTONE HI lvg/[i.Z

In 'II!

FIG. 5. Optimum histone concentration required for the maximum activity of DNA pol CYattained with varying concentrations of template primer. The activity of 2.5 units of purified enzyme was assayed as described under Materials and Methods, except that 2.2 (0). 6.7 (a). and 20 pg (0) of activated DNA were used and histone Hl was added to the reaction mixture at the indicated concentration. Filled circles indicate the activity ofthe samples, which were incubated without histone and with 20 @g/O.2 ml of activated DNA and supplemented with 2 and 4 ug of histone H 1 at the end of the incubation.

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PROTEIN

365

standard assay of the enzyme, where 20 pg/O.2 ml of activated DNA and 4 Kg/O.2 ml of histone H 1 were used. Since it has been reported that singlestranded DNA or a long stretch of the singlestranded part of activated DNA inhibits DNA pol (Yactivity probably by forming an abortive complex with the enzyme (9.18) and that the suppressed activity can be restored by some single-stranded DNA-specific binding proteins (9) we examined whether histone H 1 causes a similar effect. As shown in Fig. 7A, DNA pol (Y activity, which was assayed with a limited concentration of activated DNA (5 pg/O.2 ml), markedly decreased with an increase in the concentration of denatured DNA and reached 17% at 20 pg/O.3 ml. The suppressed activity could be restored by the addition of an appropriate concentration of histone H 1 (Fig. 7B), indicating that the observed stimulation in Fig. 5 is caused by interaction with the singlestranded part of activated DNA. Histone HI was also partially effective in the replicase assay with an excess amount of poly(dT) as template (the maximum enzyme activity was attained with 0.02 ODU of poly(dT) and additional poly (dT) was rather inhibitory): histone H 1 stimulated the replicase activity approximately twofold at an optimal concentration (8 pg/O.2 ml: 0.2 ODU of poly(dT) was used as template). However, histone HI did not stimulate primase activity at all (K. Yoshihara. unpublished results). DISCUSSION There have been many reports concerning endogenous protein factors that specifically stimulate DNA pol CYactivity. These factors can be classified into two groups: primer recognition proteins. Cl and C2, which were purified from HeLa cells ( 10) and CV- I cells derived from African green monkey kidney cells ( 1 1); and single-stranded DNA-specific binding proteins. purified from bovine thymus (7,9) and mouse ascites tumor cells

366

HIRONAKA

ET

4IL

0 ACTIVATED

DNA (pg/0.2

ml)

0.4

0.8

l/DNA

bUO.2

1.2

1.6

ml)

FIG. 6. Enzyme DNA polymerase

(0) and Double

activity attained with varying concentrations of template primer. (A) The activity of (Y (2 units) was assayed with varying concentrations of template primer in the absence the presence of histone Hl (0) at a constant histone H t/template primer ratio of 0.2 (w/w). (B) reciprocal plots of the curves in (A) are shown. Symbols used are the same as those in (A).

(8). The factors in the former group are shown to interact specifically with homologous DNA polymerase cy itself and are

;;1

1 0

,I,L 1 d 0 8 16

20

10 denatured (a/O,2

DNA ml)

histone (a/O,2

32

HI ml)

FIG. 7. Suppression of DNA pol n activity by denatured DNA and restoration by histone H 1. Enzyme assay was carried out as described under Materials and Methods (Assay I) except that the concentration of activated DNA was 5 @/0.2 ml. (A) Varying concentrations of denatured calf thymus DNA were added to the reaction mixture. (B) All samples contained 20 pgiO.2 ml of denatured calf thymus DNA in addition to 5 pg/O.2 ml of activated DNA. Varying concentrations of histone H 1 were added to the reaction mixture.

thought to allow the enzyme to slide along the template until it recognizes a primer (12). In contrast to the above factors, the stimulating proteins found in the latter group have single-strand-specific DNA-binding properties and are thought to interact stoichiometrically with a long, single-stranded stretch of activated DNA, blocking nonproductive polymerase binding sites on single-stranded DNA sequence and increasing the probability that the enzyme will bind at a correct primer site (9). The stimulatory effect elicited by the basic proteins was rather specific to DNA polymerase N species, and the optimum concentration for the stimulation paralleled the concentration of activated DNA used (Fig. 5). The latter finding indicates that a stoichiometric interaction between the basic proteins and activated DNA is the basis for the observed stimulation. Considering that single-stranded DNA strongly inhibits DNA pal (Y(Fig. 7A) and the suppressed activity can be restored by histone H 1, the observed stimulation in Fig. 5 is probably due to the interaction of histone HI with the single-stranded part of activated DNA. Thus. histone Hl and other basic proteins behave

STIMULATION

OF DNA POLYMERASE

like single-stranded DNA specific binding proteins as reported by Sapp et al. (9). Although spermine showed stimulatory effects similar to those of various basic proteins, the effect may be due to different mechanisms. F’isher and Korn (18) have reported that spermidine, a polyamine, did not decrease the aftinity of DNA pol (Yfor binding to single-stranded DNA and therefore have suggested that the stimulation by polyamine is due to modulation of enzyme-substrate complex formation at the primer terminus rather than to protection of the enzyme from forming an abortive complex with single-stranded DNA. Based on the results described above, the following arguments are feasible. First, although activated DNA is commonly used as template primer for the determination of DNA polymerase activity, the values obtained with this assay system vary markedly depending on the amount of basic protein contaminating the enzyme preparation. Thus, standar’dized use of a particular basic protein, like histone, may be necessary to compare the activities of various enzyme preparations at different purification stages. Second, since various endogenous basic proteins may show an apparent enzyme-stimulating activity through a nonspecific mechanism as observed in the present study, such activities should be classified separately from other specific regulatory factors of the enzyme. ACKNOWLEDGMENTS The authors express their gratitude to Dr. Isao Dota and other staff rnembers of Osaka City Meat Hygiene Laboratory for donation of bovine thymus and to Miss lyuko Matsuda for help with preparation of the manuscript.

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