The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase E and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-l inked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The a, 6, E and /?-like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by /?-poly(~-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase E and a 135 kDa /?-like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei a t growth arrest.

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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...