Báo cáo Y học: The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum pot

The DNA-polymerase inhibiting activity of poly(b- L -malic acid) in nuclear extract during the cell cycle of Physarum polycephalum Sabine Doerhoefer 1 , Christina Windisch 1 , Bernhard Angerer 1 , Olga I. Lavrik 2 , Bong-Seop Lee 1 and Eggehard Holler 1 1 Institut fu ¨ r Biophysik und physikalische Biochemie, Universita ¨ t, Regensburg, Germany; 2 Novosibirsk Institute of Biorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia The naturally synchronous plasmodia of myxomycetes synthesize poly(b- L -malic acid), which carries out cell-spe- cific functions. In Physarum polycephalum,poly(b- L -malate) [the salt form of poly(b- L -malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory ac tivity is cell-cycle-dependent, purified DNA polymerase a of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [ 3 H]thymidine 5¢-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To t est for the activity of endogenous DNA polymerases, DNA syn- thesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed i n S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the c ell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly( b- L -malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(b- L -malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373–380]. To explain the variation i n the cell cycle, a p eriodic competition for poly( b- L -malate) between DN A polym- erases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By c ompeti- tion they displace DNA polymerase from the complex of poly(b- L -malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(b- L -malate) is active in maintaining mitotic synchrony of plasmodia b y playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA po lymerases. Keywords: poly(malic acid); cell cycle; S-phase; DNA syn- thesis; h istones. Poly(b- L -malic acid) consists of L -malic acid units, which are covalently linked by ester bonds between the hydroxyl group and the carboxyl group in the b position, while the carboxyl group in a position points away from the polyester chain [1]. The ionized form of the polymer, poly(b- L -malate) (PMLA), amounts to high concentrations comparable to DNA in the naturally synchronous nuclei of the plasmo- dium, the giant polynuclear cell form of the slime mould Physarum polycephalum [1–3]. This organism differentiates into several cell forms during its life cycle (e.g. spores and amoebae) [4], but only the plasmodium produces poly(b- L -malic acid). In contrast to the giant cell dimensions, the billions of nuclei display cyclic events, such as mitosis and DNA replication, with a high degree o f natural synchrony. Because of these featu res, the plasmodium is suited for studying molecular biology of the cell cycle. One particular question is the organization of the catalytic competence of DNA polymerases in the context of synchrony. Poly(b- L -malate) was discovered by its activity to bind and reversibly inactivate the endogeneous DNA polymerase a [1,5]. The other replicatively active DNA polymerases (types d and e)havealsobeenshowntobindandbecome inactivated, whereas the putative repair enzyme, DNA polymerase b-like, was not inhibited [6,7]. Binding experi- ments with synthetic polyanions, which differed from PMLA in the distance between the negative charges, demonstrated that specific ity of binding is a ttributed to the particular distance b etween the n egative charges in PMLA [5]. This distance is similar to that between phosphate groups in the nucleic acid backbone, in agree- ment with the competitive binding of PMLA and DNA to the polymerases. The molecular mimicry suggested that PMLA could bind to histones and to other DNA interact- ing proteins. Indeed, large complexes of PMLA not only with DNA polymerases but also with histones and other proteins have been found under conditions close to in vivo [2,7]. The binding to histones has been further investigated by in vitro experiments [5]. If histones and DNA polymerases are together, they are prone to compete for the binding to PMLA. The periodic Correspondence to E. Holler, Institut fu ¨ r Biophysik und physikalische Biochemie der Universita ¨ t Regensburg, D-93040 Regensburg, Germany. Fax: + 49 941943 2813, Tel.: + 49 941943 3030, E-mail: eggehard.holler@biologie.uni-regensburg.de Abbreviations: AFBdCTP, exo-N-{[[((4-azido-2,3,5,6-tetra- fluorobenzylidene)hydrazino)carbonyl]butyl]carbonyl}deoxycytidine- 5¢-triphosphate; PMLA, poly(b- L -malate). Enzymes: D NA polymerase (E.C. 2.7.7.7); benzonase (E.C. 3.1.21.1). Note: a website is available at h ttp://www.biologie.uni-regensburg.de/ Biophysik (Received 8 O ctober 2001, revised 27 December 2001, accepted 7 January 2002) Eur. J. Biochem. 269, 1253–1258 (2002) Ó FEBS 2002 production of histones (or of any other PMLA-binding molecule) in S-phase could evoke a cycling of free DNA polymerases and DNA synthetic a ctivity, although the individual levels of PMLA and DNA polymerases need not vary. For a p roper understanding of the role of PMLA it was thus interesting to know its inhibitory activity over the cell cycle. Because the inhibitory activity could not be tested under in vivo conditions, experiments were carried out with nuclear extracts. The results were consistent with the assumption that PMLA was a mediator between increased concentrations of certain nuclear constituents and the competence of DNA polymerases in DNA synthesis during S-phase. MATERIALS AND METHODS Materials Microplasmodia of P. polycephalum,strainM 3 CVIII (ATCC 96951), were grown in shaken cultures at 27 °C, as described p reviously [8]. Macroplasmodia were obtained as surface cultures on fi lter paper by the fusion of mi cro- plasmodia, as described previously [9]. The mitotic stages were identified b y phase-co ntrast m icroscopy [10]. O ne gram of wet plasmodia corresponded to % 2 · 10 8 nuclei. DNA polymerase a (110 UÆmL )1 ) was purified from plasmodia as described previously [11]. DNase-I-activated salmon testis DNA for the s tandard DNA polymerase a ssay and for photoaffinity labeling was prepared as described previously [12]. Rabbit antiserum against DNA polymerases type a and type e, was prepare d with a m ixture of the purified P. polycephalum DNA polymerases [13], a nd rabbit anti- serum against DNA polymerase d by immunization with synthetic peptides of the enzyme [6]. Peroxidase-coupled anti-(rabbit IgG) Ig was purchased from Pierce. Proteinase inhibitors were used in a cocktail of the following concentrations after dilution with the e xtracts: 5 m M sodium bisulfite, 0.2 m M phenylmethanesulfonyl fluoride, 1 m M benzamidine (Sigma), 1 l M pepstatin A (Merck), 10 l M leu- peptin (Sigma), 1 mgÆmL )1 aprotinin (Merck), 10 l M tosyl- L -lysine chloromethyl ketone (Calbiochem), 100 l M pefablock S C (Merck), and 2 lgÆmL )1 E 64 (Boehringer Mannheim). For photocrosslinking, the dCTP analogue exo-N-{[[((4-azido-2,3,5,6-tetrafluorobenzylidene)hydrazi- no)carbonyl]butyl]carbonyl}deoxycytidine-5¢-triphosphate (AFBdCTP) was prepared as described previously [14], and was a gift from Safronov (Novosibirsk). Benzonase grade II (25 0 00 UÆmL )1 ) was purchased from Merck. Nonradio- active dNTPs and standard proteins for SDS/PAGE were obtained from P harmacia (Sweden). [ 3 H]dTTP (60 CiÆmmol )1 ,1Ci¼37 GBq) and [a- 32 P]dATP (3000 CiÆmmol )1 ) were purchased from Amersham. Preparation of nuclear extract Nuclei were prepared either from macroplasmodia follow- ing their third mitosis, or from microplasmodia harvested after 2 days of inoculation. The plasmodia were washed by centrifugation (500 g,10min,4°C) in cold water, suspen- ded in disruption buffer (2 g of solvent per 1 g o f wet plasmodia; 15 m M Tris/HCl pH 7.5, 5 m M EGTA, 0.5 m M CaCl 2 ,15 m M MgCl 2 , 500 m M hexylenglycol, 10% dextran, 14 m M 2-mercaptoethanol, and p rotease inhibitor c ocktail) and disrupted in a Dounce homogenizer (10–12 strokes). Nuclei were pellete d over a 25% Percoll g radient in t he above buffer, as described previously [2]. The pellet contained 2 ± 1 · 10 8 nuclei per g of wet m icroplasmodia. Nuclear extracts were prepared by incubating for 10 min on ice in an equal volume of extraction buffer (final concen- trations 50 m M Tris/HCl pH 7.5, 0.3 M KCl, 20 m M MgCl 2 , 0 .5% T riton X-100, 20% glycerol, 1 m M 2-merca- ptoethanol, protease inhibitor cocktail) and centrifugation at 700 g. The nuclear extract contained > 85% o f the total nuclear PMLA and > 75% o f t he total nuclear DNA polymerase activity in the standard assay. Results by SDS/ PAGE and Western blotting with specific antisera against DNA polymerases a and e [13], and DNA polymerase d [14] were consistent with the recovery of > 95% of DNA polymerase a and > 75% of the other DNA polymerases in the extract. Standard DNA polymerase activity and inhibition assays Total DNA polymerase activity was assayed as described previously [12]. The standard assay contained in 150 lL 50 m M 3-(N-morpholino)propanesulfonic acid/potassium salt (pH 7.5), 50 m M KCl, 10 m M MgCl 2 ,3m M EDTA, 3m M 2-mercaptoethanol, 3 3 l M each of dATP, dCTP, dGTP, 3 l M [ 3 H]dTTP (1 CiÆmmol )1 ), 20 lg DNase-I activated salmon testis DNA, 80 lg bovine serum albumin, and DNA polymerase. After a 30-min lcubation at 37 °C, 10% (v/v) saturated cold trichlo roacetic acid in water w as added and the precipitate collected on Whatman GF/C filters, which were washed with trichloroacetic acid, then with 70% (v/v) ethanol/H 2 O dried, and counted with 20% efficiency. If r equired, either 0 .4 m M spermineÆ4HCl or 2m M spermidineÆ3HCl were included to suppress binding and inhibition of polymerases by PMLA [1]. One unit of polymerase activity is equivalent to the amount of enzyme that catalyses the incorporation of 1 nmol nucleotides during 1 h. The same conditions were used in the inhibition experi- ments, except that the biogenic amines were omitted. To measure the inhibitory activity of n uclear extracts during t he cell cycle, 0.4 U of purified DNA polymerase a was present in the assay under the above standard conditions comparing the reaction rates in the presence (v i ) and the absence (reference, v o ) of nuclear extract equivalent to 1.5 · 10 5 nuclei. To account for effects of particular ingredients in the extract buffer, appropriate amounts of these reagents were added to t he reference reaction mixture. The low poly- merase activity contained in t he extract due to DNA polymerase b-like (which was not inhibited by PMLA) was measured in parallel and subtracted from the crude value for v i . The inhibitory activity is defined in terms of the reciprocal value of the inhibition constant K À 1 i ¼ [E–PMLA]/[E]Æ[PMLA]. The concentration of the poly- merase–inhibitor complex, [E–PMLA], can be expressed i n terms of [E] o ) [E]. The concentration of free polymerase, [E], and of the total polymerase [E] o is proportional to v i and v o . The expression for the relative inhibitory activity is then (v o ) v i )/v i ¼ [PMLA]/K i . Therefore, the higher the con- centration of free PMLA, and the lower the value of the inhibition constant (K i ), the higher the relative inhibitory activity (v o ) v i )/v i . In the presence of ligands that bind com- petitively to PMLA, the value of K i increases as a function 1254 S. Doerhoefer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 of rising concentrations and a ffinities of these ligands. For example, if spermine hydrochloride, which binds to PMLA, is present, the inhibition may b e totally neutralized. The reciprocal of the direct value s of the relative inh ibitory activity will be shown in the results, as they correlate directly with the degree of residual DNA polymerase activity. DNA replication of single macroplasmodia w as followed under in vivo conditions at various phases in the cell cycle. The plasmodia were grown on filter paper for a particular period of time in the cell cycle. One fourth of the plasmodium, usually 80 lg, was transferred to fresh medium containing 5 lCiÆmL )1 [methyl- 3 H]thymidine (25 CiÆmmol )1 ) and grown for 15 min at 27 °C. The remainder was allowed to grow and used as the source of further samples. The incubation was terminated by fixation in a 10-mL solution containing ice-cold 5% saturated trichoroacetic acid and 50% acetone. The samples were disrupted in a Dounce homogenizer and filtered on GF/C. After washing with trichloroacetic acid/acetone and etha- nol, the filters were dried, and the radioactivity was counted in a scintillation cocktail. Photoaffinity labeling of DNA polymerases Labeling o f DNA polymerases was carried out in a 20-lL solution containing 5 lL nuclear extract, 3–5 l M [ 32 P]dATP (3000 CiÆmmol )1 ), 125 l M AFBdCTP, 33 l M of each dGTP and dTTP, 50 m M 3-(N-morpholino)propanesulfonic acid buffer (pH 7.5), 50 m M KCl, 10 m M MgCl 2 ,3m M EDTA and 5 lg activated salmon testis DNA [7]. Escheri- chia coli DNA polymerase I served as a positive control in a parallel, but oth erwise identical, reaction mixture. In other control reactions, to exclude staining due to DNA poly- merase adenylation or phosphorylation, the photoreactive nucleotide was omitted. The polymerization reaction was carried out in the dark for 10 min at 37 °C. An aliquot was then irradiated for 2 min. Free DNA and DNA protruding from crosslinked complexes with proteins were digested with benzonase (25 U per sample) for 10–15 min a t 37 °C. The sample was heated for 3 min with Laemmli buffer [15] and e xamined b y denaturating SDS/PAGE (10% poly- acrylamide gel). Proteins were electroblotted onto Millipore Immobilon membranes [16] and visualized by autoradio- graphy (Kodak X-OMAT LS) at )70 ° (5–7 days). The identities of blotted proteins were verified by immunostain- ing of the same membranes. Intensities of bands were quantified with a Boehringer Mannheim Lumi Imager TM . The intensity of labeled E. coli DNA polymerase I in the same gel served as a reference. RESULTS Finding optimal assay conditions to measure the inhibitory activity in nuclear extracts In previous analytical and preparative experiments, it has been shown that PMLA was the constituent that s pecific- ally inhibited replicative DNA polymerases in nuclear extracts [1,2,7]. In the present study, we measured the inhibitory activity of PMLA using purified DNA poly- merase a of P. polycephalum as an added reporter. To find the optimal assay conditions, t he extracts were prepared from the nuclei of microplasmodia that naturally included all phases o f the c ell cycle. As considered in Materials and methods, t he degree of inhibition depends on both the concentration of free PMLA and the inhibition constant. This parameter reflected the affinity of the polymerase and both the affinity and concentration of competing ligands for binding to the polyanion. In the (added) nuclear extract, such ligands were histones, and probably other DNA- binding proteins [2]. T o obtain an optimal response by the reporter polymerase to a varying i nhibitory activity in the nuclear extract, the amounts o f the added polymerase a nd extract had to be optimized. To this end, the titration of a fixed amount of nuclear extract, c orresponding to 1.5 · 10 5 nuclei, was performed in the first experiment (Fig. 1). In the beginning of the titration, the polymerase activity remain ed suppressed until the inhibitory activity was neutralized by an amount of 0.38 ± 0.03 U of the reporter DNA polymerase (the arrow in Fig. 1). During continued addi- tion of the polymerase, the enzyme a ctivity increased in parallel with the activity of the control experiment in the absence of extract. An a mount of 0.4 U of the r eporter DNA polymerase, close to the neutralization point, was chosen for the measurement o f the inhibitory activity during t he cell cycle. We have previously shown that the inhibitory activity of purified PMLA is neutralized in the presence 0.4 m M spermine hydrochloride [1]. To confirm t hat PMLA was the only inhibitor of DNA polymerases in the extract above, we measured the polymerase activity in the presence of added spermine hydrochloride. A value of 1.2 ± 0.03 U (five measurements) was ob se rved and r eferred t o the endogenous DNA polymerases. The experiment was repea- ted with the extract containing in addition 0.4 U reporter DNA polymerase a. A n amount of 1.6 ± 0.03 U was measured in this case (five measurements). The difference of 0.4 ± 0.04 U was in agreement with the added 0.4 U. The same results were obtained when nuclear extracts in the S-phase and in G2-phase were compared. The agreement was consistent with the assumption that the inhibitory activity was a property of PMLA in the extract. Fig. 1. The inhibition of purified DNA polymerase a by po ly(b- L - malate) in the nuclear extract. (d) The activity was measured as a function of added a mounts of DNA polymerase a in the standard DNA polymerase assay that contained the extract of 2 · 10 6 nuclei isolated from microplasmodia. (j) The control in the absence of the nuclear extract containing an equivalent of the ingredients in the nuclear e xtraction buffer. The arrow refers to the equivalence of the added DNA p olymerase activity and t he inhibitory activity. Ó FEBS 2002 Poly(b- L -malate) mediated DNA polymerase activity (Eur. J. Biochem. 269) 1255 The inhibitory activity during the cell cycle The inhibitory activity during the cell cycle was measured in the p resence of 0.4 U of (added) purified DNA polymerase a and the extract of 1.5 · 10 5 nuclei. The dependence is shown in Fig. 2A in terms of the reciprocal values, corresponding to the residual DNA polymerase activity. The m aximum at 1 h after m itosis corresponded t o a minimum in inhibitory activity and a maximum i n t he residual polymerase a ctivity (63% of the reference activity). After 2 h following mitosis and during the remainder of t he cell cycle, the polymerase activity approached a basal level of 10–20% of the reference activity. The activity of DNA polymerases measured by photo affinity labeling According to Fig. 2A, the inhibitory activity in the nuclear extract s howed a minimum between 0 and 2 h after mitosis (Fig. 2 A). It was of interest whether this interval coincided with some endogenous residual activity of the DNA polymerases in the n uclear extract (DNA polymerase a not added). Because t he (residual) activity of the e ndo- genous DNA polymerases was too low to be detected with the standard assay, we introduced a highly sensitive technique of affinity photo crosslinking [7]. Briefly, the enzymatically active DNA polymerase c atalysed the primer elongation w ith r adioactively labeled nucleotides o f h igh specific radioactivity. Then , the e longated primers w ere photo crosslinked to the active polymerases within the elongation complex. The amount of radioactivity covalently attached to DNA polymerases was an i ndicator of the polymerase activity and was measured after SDS/PAGE by autoradiography. S eparate results are shown in Fig. 2C for DNA polymerase e and for DNA polymerases of type a, type-b-like, and type d in Fig. 2B, which were not resolved from each other. DNA polymerase e showed the highest activity during the first hour after mitosis. Then the activity Fig. 2. DNA polymerase activitiy during the cell cycle of macroplasmodia. All graphs are drawn to the same scale to facilitate comparison. In this scale, the measured value at 0.7 h in the nuclear division c ycle is arbitrarily set equal to one u nit in each panel. M denotes m itosis. (A) The reciprocal inhibitory activity v i (v o ) v i ) )1 (see text) calculated from values of the r esidual polymerase activity (v i ) and the reference activity (v o )ofadded0.4 U of purified DNA po lymerase a in the standard DNA polymerase assay with (v i ) and without (v o ) nuclear extract. The extract of 2 · 10 6 nuclei was prepared from macroplasmodia at various times during the cell cycle. Bars refer to standard deviations (three determinations). One unit on scale refers to 1.64 U of th e recip rocal in hibitory activity. (B) Activity o f endo geno us D NA polymerases a, d,andb-like in the nuclea r e xtract, measured in arbitrary units (staining intensity) by the highly sensitive technique o f photoaffinity labeling. Single types of DNA polymerases could n ot be resolved. Bars refer to standard deviations (three determinations). (C) Activity of endogenous DNA polymerase e, measured in parallel with t he DNA polymerases in panel B. One u nit o n scale compares to half a unit in (B). (D) DNA synthesizing activity o f plasmodia at various times in the cell cycle. The incorporation of radioactivity into acid precipitable material has been measured during a brief exposure to [methyl- 3 H]thymidine. One u nit on scale refers to 6 Bq [ 3 H]TMP incorporated. (E) The activity of endogenous D NA polymerases in the extract o f 2 · 10 8 nuclei at various times during the cell cycle. One unit on scale refers to 90 U of DNA po lymerase activity (see Materials an d metho ds). T he activity was measured i n the presence of added 2 m M spermidineÆ3HCl (?) to neutralize t he inhibitory activity o f PMLA. I n the absence o f spermidineÆ3HCl, activities of DNA polymerase, except o f DNA polymerase b-like, are inhibited by PMLA contained in the extracts. 1256 S. Doerhoefer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 decreased and fell to a basal level 2 h after mitosis. The dependence was sim ilar f or the unresolved DNA poly- merases. The high basal level was explained by the contribution of DNA polymerase b-like, which was not inhibited by PMLA. The results show that the minimum in the inhibitory activity corresponded with a maximum of DNA polymerase activity in the cell cycle. The in vivo activity of DNA polymerases The cell cycle depen dence of DNA polymerase activity w as followed under in vivo conditions. The incorporation of radioactivity into DNA was determined following a brief exposure to [methyl- 3 H]thymidine. The results in Fig. 2D show a maximum in activity of DNA synthesis a t 1 h after mitosis, followed by a decrease approaching a basal level at 2 h after mitosis. Thus, the in vivo and in vitro activities of DNA synthesis c orresponded with the minimum of the inhibitory activity in the cell cycle. The activity of DNA polymerases in the nuclear extracts after neutralization of the inhibitory activity by spermidine hydrochloride Although the appearance of an activity peak of DNA polymerases was consistent with the minimum in the inhibitory activity of PMLA, an additional periodic variation in the intrinsic activities of the endogenous DNA polymerases was not excluded. It has b een shown that biogenic polyamines bind to PMLA and n eutralize its inhibitory activity against DNA polymerases [1]. This finding allowed us to examine whether the intrinsic DNA polymerase activity of the extract varied during the cell cycle or whether it w as totally modulated by the degree of complex formation of DNA polymerases with PMLA. The circles in Fig. 2E show the DNA polymerase activity of nuclear extract in the standard assay in the case when spermidine hydrochloride was not present. This a ctivity referred to DNA polymerase b-like, which was not inhibited by PMLA [1] and thus did not show a cell cycle dependence. The other DNA polymerases displayed no m easurable activity in the standard assay due to the strong inhibition by PMLA (see also above). The squares i n Fig. 2E refer to the addition of spermidine hydrochloride. The activities of the DNA polymerases were derepressed, because the inhibitory activity was neutralized. The data show the superpositions for DNA polymerases a–e. Importantly, a cell cycle dependence was not indicated. The variations observed in Fig. 2A–C were explained by the change in the inhibitory activity of PMLA during the cell cycle. DISCUSSION Myxomycetes comprise a large family of organisms that typically gen erate a plasmodium, a polynucleated giant cell among other cell forms in the life c ycle [4]. Interestingly, the billions of nuclei in these syncytia participate with high degree of synchrony in the division cycle. All of the myxomycetes species so far examined contained PMLA in the plamodia. The PMLA level in the nuclei is high and comparable to that of chromosomal DNA [1,3]. It remains constant during the cell cycle, and PMLA synthesized in excess amounts is secreted into the culture medium. In contrast to varying degrees of PMLA synthesis, the content in the nuclei is conserved among different species (Karl, M., Anderson, R. W. & Holler, E., unpublished data). The various observations suggest that PMLA may play a role in many biological functions. One function has been attributed to the induction of sporulation of P. polycephalum [17] and another to the carriage and s torage of DNA polymerases, histones, and other nuclear proteins in the plasmodium [2,18]. In connection with the role as a carrier and storage function, the inhibitory activity of PMLA towards the replicative DNA polymerases was of interest [ 1,6,7]. The coupling of the carrier/storage function and the inhibition of DNA synthesis suggested an effect on the availability of DNA polymerase activity during t he cell cycle. Our r esults showed an inverse relation of the inhibitory activity with the activity of the endogenous DNA polymerases in the extract as well as with the DNA synthetic activity (S-phase) in living plasmodia. While the DNA synthetic a ctivity in t he nuclear extract was periodic with a maximum in S-phase, t he activities of the DNA polymerases in the standard assay were constant after n eutralization of PMLA. The cell cycle- independent biosynthesis and activity of DNA polymerase a has been described by Western blotting and a ctivity gel analysis [19]. The findings extend t he storage/carrier role of PMLA to a controller function of the catalytic competence of DNA polymerases. The results in Fig. 2 E revealed that the activities of DNA polymerases on their own did not vary and that they were inhibited by complex formation with PMLA. The inhibition would be permanent unless the complexes dissociated in S-phase. The dissociation could be principally controlled by a periodical decrease in the level of PMLA. However, the nu clear content of PMLA has been shown to be constant over the cell cycle [3]. As a carrier, PMLA binds also histones and other nuclear proteins [2]. Core histones, for example, are heavily synthesized in S-phase [20], and are likely to compete with DNA poly- merases for the binding of PMLA. Once free, DNA polymerase is c ompetent for DNA synthesis. However, the i dentity of these effectors i s still unclear. We f avor histones, because they are depleted from the PMLA complexes by forming nucleosomes, when their synthesis ceases at the end of S-phase. This would explain the e nd of the activity period of DNA polymerases. Instead of assuming a neutralization of the inhibitory a ctivity, the DNA polymerases could bind effectors, which induce the release of polymalate from t he complex. While this mech- anism is principally possible, it is not supported by experimental evidence. PMLA binds to DNA polymerases competitively with DNA [1], and such factors would also inhibit the binding of template-primer DNA and thus the polymerase activity. Although t he inhibitory activity of PMLA and its cycling has been established i n the nuclear extract, it is speculated th at a s imilar mechanism exists in t he nuclei of plasmodia. A major reason for this assumption is the finding that PMLA forms complexes with DNA polymerases, histones a nd other nuclear proteins under conditions close to in vivo [2,7]. The PMLA-dependent cycling of DNA polymerases does not account for the timing of DNA r eplication. It also does not take into account the periodic synthesis of factors such as the proliferating-cell nuclear antigen (PCNA) and Ó FEBS 2002 Poly(b- L -malate) mediated DNA polymerase activity (Eur. J. Biochem. 269) 1257 replication factor C ( RF-C) [7], but merely links the catalytic competence of DNA synthesizing polypeptides to the S- phase. These factors are recognized only with specialized template-primers but not with activated salmon testis DNA, as used here. 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The activity of DNA polymerases in the nuclear extracts after neutralization of the inhibitory activity by spermidine hydrochloride Although. 1255 The inhibitory activity during the cell cycle The inhibitory activity during the cell cycle was measured in the p resence of 0.4 U of (added) purified DNA polymerase a and the extract of 1.5. the minimum in the inhibitory activity corresponded with a maximum of DNA polymerase activity in the cell cycle. The in vivo activity of DNA polymerases The cell cycle depen dence of DNA polymerase