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

Eur. J. Biochem. 269, 1253–1258 (2002) (cid:211) FEBS 2002

The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarumpolycephalum

Sabine Doerhoefer1, Christina Windisch1, Bernhard Angerer1, Olga I. Lavrik2, Bong-Seop Lee1 and Eggehard Holler1 1Institut fu¨r Biophysik und physikalische Biochemie, Universita¨t, Regensburg, Germany; 2Novosibirsk Institute of Biorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia

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 in the cell cycle, a periodic competition for poly(b-L-malate) between DNA polym- erases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competi- 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 by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.

DNA replication, with a high degree of natural synchrony. Because of these features, 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-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

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) have also been shown to bind and become 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 specificity of binding is attributed to the particular distance between the negative 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].

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: DNA polymerase (E.C. 2.7.7.7); benzonase (E.C. 3.1.21.1). Note: a website is available at http://www.biologie.uni-regensburg.de/ Biophysik (Received 8 October 2001, revised 27 December 2001, accepted 7 January 2002)

Keywords: poly(malic acid); cell cycle; S-phase; DNA syn- thesis; histones. 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 activity 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 [3H]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 test for the activity of endogenous DNA polymerases, DNA syn- thesis was followed by the highly sensitive photoa(cid:129)nity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine

If histones and DNA polymerases are together, they are prone to compete for the binding to PMLA. The periodic

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production of histones (or of any other PMLA-binding molecule) in S-phase could evoke a cycling of free DNA polymerases and DNA synthetic activity, although the individual levels of PMLA and DNA polymerases need not vary. For a proper 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.

M A T E R I A L S A N D M E T H O D S

and disrupted in a Dounce homogenizer (10–12 strokes). Nuclei were pelleted over a 25% Percoll gradient in the above buffer, as described previously [2]. The pellet contained 2 (cid:139) 1 · 108 nuclei per g of wet microplasmodia. Nuclear extracts were prepared by incubating for 10 min on ice in an equal volume of extraction buffer (final concen- trations 50 mM Tris/HCl pH 7.5, 0.3 M KCl, 20 mM MgCl2, 0.5% Triton X-100, 20% glycerol, 1 mM 2-merca- ptoethanol, protease inhibitor cocktail) and centrifugation at 700 g. The nuclear extract contained > 85% of the total nuclear PMLA and > 75% of the 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. Materials

Total DNA polymerase activity was assayed as described previously [12]. The standard assay contained in 150 lL 50 mM 3-(N-morpholino)propanesulfonic acid/potassium salt (pH 7.5), 50 mM KCl, 10 mM MgCl2, 3 mM EDTA, 3 mM 2-mercaptoethanol, 33 lM each of dATP, dCTP, dGTP, 3 lM [3H]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 (cid:176)C, 10% (v/v) saturated cold trichloroacetic acid in water was added and the precipitate collected on Whatman GF/C filters, which were washed with trichloroacetic acid, then with 70% (v/v) ethanol/H2O dried, and counted with 20% efficiency. If required, either 0.4 mM spermineÆ4HCl or 2 mM 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.

Standard DNA polymerase activity and inhibition assays

i

and Microplasmodia of P. polycephalum, strain M3CVIII (ATCC 96951), were grown in shaken cultures at 27 (cid:176)C, as described previously [8]. Macroplasmodia were obtained as surface cultures on filter paper by the fusion of micro- plasmodia, as described previously [9]. The mitotic stages were identified by phase-contrast microscopy [10]. One gram of wet plasmodia corresponded to (cid:25) 2 · 108 nuclei. DNA polymerase a (110 UÆmL)1) was purified from plasmodia as described previously [11]. DNase-I-activated salmon testis DNA for the standard DNA polymerase assay and for photoaffinity labeling was prepared as described previously [12]. Rabbit antiserum against DNA polymerases type a and type e, was prepared with a mixture of the purified P. polycephalum DNA polymerases [13], and 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 extracts: 5 mM sodium bisulfite, 0.2 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine (Sigma), 1 lM pepstatin A (Merck), 10 lM leu- peptin (Sigma), 1 mgÆmL)1 aprotinin (Merck), 10 lM tosyl-L-lysine chloromethyl ketone (Calbiochem), 100 lM pefablock SC (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 (25 000 UÆmL)1) was purchased from Merck. Nonradio- active dNTPs and standard proteins for SDS/PAGE [3H]dTTP were obtained from Pharmacia (Sweden). (60 CiÆmmol)1, [a-32P]dATP 1 Ci (cid:136) 37 GBq) (3000 CiÆmmol)1) were purchased from Amersham.

Preparation of nuclear extract

The same conditions were used in the inhibition experi- ments, except that the biogenic amines were omitted. To measure the inhibitory activity of nuclear extracts during the 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 (vi) and the absence (reference, vo) of nuclear extract equivalent to 1.5 · 105 nuclei. To account for effects of particular ingredients in the extract buffer, appropriate amounts of these reagents were added to the reference reaction mixture. The low poly- merase activity contained in the extract due to DNA polymerase b-like (which was not inhibited by PMLA) was measured in parallel and subtracted from the crude value for vi. The inhibitory activity is defined in terms of the reciprocal value of the inhibition constant K (cid:255) 1 (cid:136) [E–PMLA]/[E]Æ[PMLA]. The concentration of the poly- merase–inhibitor complex, [E–PMLA], can be expressed in terms of [E]o ) [E]. The concentration of free polymerase, [E], and of the total polymerase [E]o is proportional to vi and vo. The expression for the relative inhibitory activity is then (vo ) vi)/vi (cid:136) [PMLA]/Ki. Therefore, the higher the con- centration of free PMLA, and the lower the value of the inhibition constant (Ki), the higher the relative inhibitory activity (vo ) vi)/vi. In the presence of ligands that bind com- petitively to PMLA, the value of Ki increases as a function

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, 10 min, 4 (cid:176)C) in cold water, suspen- ded in disruption buffer (2 g of solvent per 1 g of wet plasmodia; 15 mM Tris/HCl pH 7.5, 5 mM EGTA, 0.5 mM CaCl2, 15 mM MgCl2, 500 mM hexylenglycol, 10% dextran, 14 mM 2-mercaptoethanol, and protease inhibitor cocktail)

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of rising concentrations and affinities of these ligands. For example, if spermine hydrochloride, which binds to PMLA, is present, the inhibition may be totally neutralized. The reciprocal of the direct values of the relative inhibitory activity will be shown in the results, as they correlate directly with the degree of residual DNA polymerase activity.

time in the cell cycle. One fourth of

Fig. 1. The inhibition of purified DNA polymerase a by poly(b-L- malate) in the nuclear extract. (d) The activity was measured as a function of added amounts of DNA polymerase a in the standard DNA polymerase assay that contained the extract of 2 · 106 nuclei isolated from microplasmodia. (j) The control in the absence of the nuclear extract containing an equivalent of the ingredients in the nuclear extraction buffer. The arrow refers to the equivalence of the added DNA polymerase activity and the inhibitory activity.

DNA replication of single macroplasmodia was followed under in vivo conditions at various phases in the cell cycle. The plasmodia were grown on filter paper for a particular period of the plasmodium, usually 80 lg, was transferred to fresh medium containing 5 lCiÆmL)1 [methyl-3H]thymidine (25 CiÆmmol)1) and grown for 15 min at 27 (cid:176)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.

all phases of the cell cycle. As considered in Materials and methods, the 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]. To obtain an optimal response by the reporter polymerase to a varying inhibitory activity in the nuclear extract, the amounts of the added polymerase and extract had to be optimized. To this end, the titration of a fixed amount of nuclear extract, corresponding to 1.5 · 105 nuclei, was performed in the first experiment (Fig. 1). In the beginning of the titration, the polymerase activity remained suppressed until the inhibitory activity was neutralized by an amount of 0.38 (cid:139) 0.03 U of the reporter DNA polymerase (the arrow in Fig. 1). During continued addi- tion of the polymerase, the enzyme activity increased in parallel with the activity of the control experiment in the absence of extract. An amount of 0.4 U of the reporter DNA polymerase, close to the neutralization point, was chosen for the measurement of the inhibitory activity during the cell cycle.

Labeling of DNA polymerases was carried out in a 20-lL solution containing 5 lL nuclear extract, 3–5 lM [32P]dATP (3000 CiÆmmol)1), 125 lM AFBdCTP, 33 lM of each dGTP and dTTP, 50 mM 3-(N-morpholino)propanesulfonic acid buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 3 mM EDTA and 5 lg activated salmon testis DNA [7]. Escheri- chia coli DNA polymerase I served as a positive control in a parallel, but otherwise 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 (cid:176)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 at 37 (cid:176)C. The sample was heated for 3 min with Laemmli buffer [15] and examined by 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(cid:176) (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 ImagerTM. The intensity of labeled E. coli DNA polymerase I in the same gel served as a reference.

Photoaffinity labeling of DNA polymerases

R E S U L T S

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 specific- 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, the extracts were prepared from the nuclei of microplasmodia that naturally included We have previously shown that the inhibitory activity of purified PMLA is neutralized in the presence 0.4 mM spermine hydrochloride [1]. To confirm that 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 (cid:139) 0.03 U (five measurements) was observed and referred to the endogenous DNA polymerases. The experiment was repea- ted with the extract containing in addition 0.4 U reporter DNA polymerase a. An amount of 1.6 (cid:139) 0.03 U was measured in this case (five measurements). The difference of 0.4 (cid:139) 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.

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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 cycle is arbitrarily set equal to one unit in each panel. M denotes mitosis. (A) The reciprocal inhibitory activity vi(vo ) vi))1 (see text) calculated from values of the residual polymerase activity (vi) and the reference activity (vo) of added 0.4 U of purified DNA polymerase a in the standard DNA polymerase assay with (vi) and without (vo) nuclear extract. The extract of 2 · 106 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 the reciprocal inhibitory activity. (B) Activity of endogenous DNA polymerases a, d, and b-like in the nuclear extract, measured in arbitrary units (staining intensity) by the highly sensitive technique of photoa(cid:129)nity labeling. Single types of DNA polymerases could not be resolved. Bars refer to standard deviations (three determinations). (C) Activity of endogenous DNA polymerase e, measured in parallel with the DNA polymerases in panel B. One unit on scale compares to half a unit in (B). (D) DNA synthesizing activity of 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-3H]thymidine. One unit on scale refers to 6 Bq [3H]TMP incorporated. (E) The activity of endogenous DNA polymerases in the extract of 2 · 108 nuclei at various times during the cell cycle. One unit on scale refers to 90 U of DNA polymerase activity (see Materials and methods). The activity was measured in the presence of added 2 mM spermidineÆ3HCl (?) to neutralize the inhibitory activity of PMLA. In the absence of spermidineÆ3HCl, activities of DNA polymerase, except of DNA polymerase b-like, are inhibited by PMLA contained in the extracts.

The inhibitory activity during the cell cycle

The activity of DNA polymerases measured by photo affinity labeling

The inhibitory activity during the cell cycle was measured in the presence of 0.4 U of (added) purified DNA polymerase a and the extract of 1.5 · 105 nuclei. The dependence is shown in Fig. 2A in terms of the reciprocal values, corresponding to the residual DNA polymerase activity. The maximum at 1 h after mitosis corresponded to a minimum in inhibitory activity and a maximum in the residual polymerase activity (63% of the reference activity). After 2 h following mitosis and during the remainder of the cell cycle, the polymerase activity approached a basal level of 10–20% of the reference activity.

(Fig. 2A). It was of interest whether this interval coincided with some endogenous residual activity of the DNA polymerases in the nuclear extract (DNA polymerase a not added). Because the (residual) activity of the endo- 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 catalysed the primer elongation with radioactively labeled nucleotides of high specific radioactivity. Then, the elongated primers were photo crosslinked to the active polymerases within the elongation complex. The amount of radioactivity covalently attached to DNA polymerases was an indicator of the polymerase activity and was measured after SDS/PAGE by autoradiography. Separate 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 According to Fig. 2A, the inhibitory activity in the nuclear extract showed a minimum between 0 and 2 h after mitosis

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decreased and fell to a basal level 2 h after mitosis. The dependence was similar for 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.

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 storage of DNA polymerases, histones, and other nuclear proteins in the plasmodium [2,18]. The invivoactivity of DNA polymerases

The cell cycle dependence of DNA polymerase activity was followed under in vivo conditions. The incorporation of radioactivity into DNA was determined following a brief exposure to [methyl-3H]thymidine. The results in Fig. 2D show a maximum in activity of DNA synthesis at 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 corresponded 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

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 the cell cycle. Our results 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 activity in the nuclear extract was periodic with a maximum in S-phase, the activities of the DNA polymerases in the standard assay were constant after neutralization of PMLA. The cell cycle- independent biosynthesis and activity of DNA polymerase a has been described by Western blotting and activity gel analysis [19].

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 been shown that biogenic polyamines bind to PMLA and neutralize 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 was 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 activity 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 measurable activity in the standard assay due to the strong inhibition by PMLA (see also above). The squares in 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.

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D I S C U S S I O N

The findings extend the storage/carrier role of PMLA to a controller function of the catalytic competence of DNA polymerases. The results in Fig. 2E 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 nuclear 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 competent for DNA synthesis. However, the identity of these effectors is still unclear. We favor 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 end of the activity period of DNA polymerases. Instead of assuming a neutralization of the inhibitory activity, the DNA polymerases could bind effectors, which induce the release of polymalate from the complex. While this mech- is not supported by anism is principally possible, 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 the inhibitory activity of PMLA and its cycling has been established in the nuclear extract, it is speculated that a similar mechanism exists in the nuclei of plasmodia. A major reason for this assumption is the finding that PMLA forms complexes with DNA polymerases, histones and other nuclear proteins under conditions close to in vivo [2,7].

Myxomycetes comprise a large family of organisms that typically generate a plasmodium, a polynucleated giant cell among other cell forms in the life cycle [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 The PMLA-dependent cycling of DNA polymerases does not account for the timing of DNA replication. It also does not take into account the periodic synthesis of factors such as the proliferating-cell nuclear antigen (PCNA) and

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8. Daniel, J.W. & Baldwin, H.H. (1964) Methods of culture for

plasmodial myxomycetes. Methods Cell Physiol. 1, 9–14.

9. Nygaard, O.P. & Guttes, S.R.H.P. (1960) Nucleic acid metabolism in a slime mold with synchronous mitosis. Biochim. Biophys. Acta 38, 298–306.

10. Mohberg, J. (1982) Recognition of mitosis. In Cell Biology of Physarum and Didymium (Aldrich, H.C. & Daniel, J.W., eds), pp. 273–276. Academic Press, New York, NY.

11. Weber, C., Fischer, H. & Holler, E. (1988) Purification and characterization of DNA polymerase a from plasmodia of Physarum polycephalum. Eur. J. Biochem. 176, 199–206.

synchrony by coordinating the

12. Holler, E., Fischer, H., Weber, C., Stopper, H., Steger, H. & Simek, H. (1987) A DNA polymerase with unusual properties from the slime mold Physarum polycephalum. Eur. J. Biochem. 163, 397–405.

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. Because PMLA is only found in the multinucleated plasmodia and not in the mononucleated amoebae, it is speculated that the periodic change of the inhibitory activity is involved in the maintenance of the plasmodial catalytic competence of DNA polymerases throughout the giant cell. We are currently investigating the transfer of PMLA between the nuclei, and the competitive exchange of DNA polymerases and effector proteins.

R E F E R E N C E S

13. Achhammer, G., Angerer, B., Windisch, C., Uhl, A. & Holler, E. (1992) DNA Polymerase a-primase complexes of Physarum polycephalum. Cell. Biol. Int. Reports 16, 1047–1053.

1. Fischer, H., Erdmann, S. & Holler, E. (1989) An unusual poly- anion from Physarum. polycephalum that inhibits homologous DNA polymerase a in vitro. Biochemistry 28, 5219–5226.

14. Safronov, I.V., Sherbick, N.V., Khodyreva, S.N., Wlassoff, W.A., Dobrikov, M.I., Shishkin, G.V. & Lavrik, O.I. (1997) New photoreactive N4-substituted dCTP analogues: preparation, photochemical characteristics, and substrate properties in HIV-1 reverse transcriptase-catalyzed DNA synthesis. Russ. J. Bioorg. Chem. 23, 576–585.

2. Angerer, B. & Holler, E. (1995) Large complexes of b-poly (L-malate) with DNA polymerase a, histones, and other proteins in nuclei of growing plasmodia of Physarum polycephalum. Bio- chemistry 34, 14741–14751.

15. Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

3. Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumu- lation and homeostasis of the unusual polymer b-poly (L-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell. Biol. 70, 373–380.

16. Towbin, H., Staehlin, T. & Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76, 4350–4354.

4. Burland, T.G., Solnica, K.L., Bailey, J., Cunningham, D.B. & Dove, W.F. (1993) Patterns of inheritance, development and the mitotic cycle in the protist Physarum polycephalum. Adv. Microb. Physiol. 35, 1–69.

17. Renzel, S., Esselborn, S., Sauer, H.W. & Hildebrandt, A. (2000) Calcium and Malate are sporulation-promoting factors of Physarum polycephalum. J. Bacteriol. 182, 6900–6905.

5. Holler, E., Achhammer, G., Angerer, B., Gantz, B., Hambach, C., Reisner, H., Seidel, B., Weber, C., Windisch, C., Braud, C., Guerin, P. & Vert, M. (1992) Specific inhibition of Physarum polycephalum DNA-polymerase-a-primase by poly (L-malate) and related polyanions. Eur. J. Biochem. 206, 1–6.

18. Rathberger, K., Reisner, H.W.B., Molitoris, H.-P. & Holler, E. (1999) Comparative synthesis and hydrolytic degradation of poly (L-malate) by myxomycetes and fungi. Mycol. Res. 103, 513–520. 19. MacNicol, A.M., Banks, G.R. & Cox, R.A. (1987) Biosynthesis and activity of DNA polymerase throughout the mitotic cycle of Physarum polycephalum. FEBS Lett. 221, 48–54.

20. Loidl, P. & Gro¨ bner, P. (1987) Histone synthesis during the cell cycle of Physarum polycephalum. Synthesis of different histone species is not under a common regulatory control. J. Biol. Chem. 262, 10195–10199.

6. Achhammer, G., Winkler, A., Angerer, B. & Holler, E. (1995) DNA polymerase d of Physarum polycephalum. Curr. Genet. 28, 534–545. 7. Doerhoefer, S., Khodyreva, S., Safronov, I.V., Wlassoff, W.A., Anarbaev, R., Lavrik, O.I. & Holler, E. (1998) Molecular consituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoa(cid:129)nity labelling. Microbiology 144, 3181–3193.