Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-a-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-a-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(viny1 sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (A') interspaced between them. Poly(L-malate) ( N = 5 ) and pOly(D,L-malate) ( N = 5 ) are the most efficient inhibitors, followed by poly(L-aspartate) ( N = 6), poly(acry1ate) ( N = 3), poly(Lglutamate) ( N = 8), poly(viny1 sulfate) ( N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-a-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-a-primase in the G 2 phase and M phase of the plasmodial cell cycle.Plasmodia1 cells of P. polycephalum have been of particular interest to cell biologists because of their giant multinucleated forms and their high synchrony in nuclear division [l]. In some instances, they resemble syncytically organized cells found during early stages of embryogenesis. P. polycephalum develops DNA polymerase a and newly replicated DNA may interact during the cell cycle competitively in such a way that DNA polymerase a is active during the S phase and inactive during the G2 phase. In this hypothesis, the initiation of histone synthesis marks the beginning of the S phase ([5], for a recent review about histones see [6]). The newly synthesized histones displace DNA polymerase CI from poly(L-malate) by competition. The released polymerase becomes involved in DNA replication until histone synthesis ceases, and histones are consumed in the energetically highly favorable formation of nucleosomes by newly replicated DNA. Free poly(L-malate) reassociates with DNA polymerase a at the onset of the G2 phase and thereby terminates DNA synthesis. Thus, poly(~-malate) may function in stock piling inactive DNA polymerase during the G 2 phase and M phase of the cell cycle. In the intact cell, DNA polymerase a is associated with DNA primase. Activities of these enzymes function coordinately during the start of DNA synthesis at replication origins and during initiation and elongation of Okazaki pieces (for a recent review see [...

β‐Poly‐l‐malate (PMA) is a highly soluble polyanion specifically synthesized during the plasmodial stage of the life cycle in Physarum polycephalum. The polymer partitions in a growth‐dependent fashion between cell nuclei, the cytoplasm and the culture medium, where it is slowly hydrolyzed. It strongly interacts with certain soluble nuclear proteins suggesting the function as a mobile matrix involved in stock‐piling of such molecules and required in the synchronization of the nuclear cell division typical for plasmodia. It is synthesized continuously over the cell cycle during growth involving a PMA polymerase that seems genetically related to the enzyme catalyzing in certain bacteria the polymerization of poly‐β‐hydroxybutyrate.

DNA polymerase alpha and DNA polymerase alpha--primase complex of Physarum polycephalum were purified by rapid methods, and antibodies were raised against the complex. In crude extracts, immune-reactive polypeptides of 220 kDa, 180 kDa, 150 kDa, 140 kDa, 110 kDa, 86 kDa, 57 kDa and 52 kDa were identified. The structural relationships between the 220 kDa, 110 kDa and 140 kDa (the most abundant form) was investigated by peptide mapping. The 140 kDa form was active DNA polymerase alpha. The 57 kDa and the 52 kDa polypeptides were identified as primase subunits by auto-catalytic labelling. In amoebae, the immune-reactive 140 kDa polypeptide was replaced by a 135 kDa active DNA polymerase alpha.

The naturally synchronous plasmodia of myxomycetes synthesize poly(b-L-malic acid), which carries out cell-specific 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 [ 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 test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity 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 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 polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition 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.Keywords: poly(malic acid); cell cycle; S-phase; DNA synthesis; histones.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 plasmodium, the giant polynuclear cell form of the slime mould Physarum polycephalum [1][2][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 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...

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