DNA polymerase α-primase complex of Physarum polycephalum

Achhammer, Gunthar; Angerer, Bernhard; Windisch, C; Uhl, A.; Holler, Eggehard

doi:10.1016/s0309-1651(05)80028-1

Cited by 7 publications

(19 citation statements)

References 10 publications

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“…The polyanion consists of units of l ‐malate, which are esterified between the hydroxyl group and the β‐carboxyl group to form a linear polyester of a number‐averaged molecular mass of 50 000. It binds replicative DNA polymerases, histones and other nuclear proteins reversibly [5,7–10]. Polymalate synthetase has been studied in vivo [11], but neither the locus nor the proteins involved in the synthesis could be identified.…”

mentioning

confidence: 99%

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Karl¹,

Gasselmaier²,

Krieg

et al. 2003

European Journal of Biochemistry

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The nuclei in the plasmodium of Physarum polycephalum, as of other myxomycetes, contain high amounts of polymalate, which has been proposed to function as a scaffold for the carriage and storage of several DNA-binding proteins [Angerer, B. and Holler, E. (1995) Biochemistry 34, 14741-14751]. By delivering fluorescence-labeled polymalate into a growing plasmodium by injection, we observed microscopic staining of nuclei in agreement with the proposed function. The fluorescence intensity was highest during the reconstruction phase of the nuclei. To examine whether the delivery was under the control of polymalatase or related proteins [Karl, M. & Holler, E. (1998) Eur. J. Biochem. 251, 405-412], the cellular distribution of these proteins was also examined by staining with antibodies against polymalatase. Double-stained plasmodia revealed a fluorescent halo around each fluorescent nucleus during the reconsititution. Fluorescent nuclei were not observed when the hydroxyl terminus of polymalate, known to be essential for the binding of polymalatase, was blocked by labeling with fluorescein-5-isothiocyanate. By immune precipitation, it was shown that polymalate and polymalatase or related proteins were in the precipitate. It is concluded that polymalate is delivered to the surface of nuclei in the complex with polymalatase or related proteins. The complex dissociates, and polymalate translocates into the nucleus, while polymalatase or related proteins remain at the surface.Keywords: Physarum polycephalum; plasmodium; polymalic acid; polymalatase; reconstituting nuclei.Physarum polycephalum is a well characterized member of the plasmodial slime molds (myxomycetes) that typically have a life cycle involving haploid (spores, amoebae) and diploid (plasmodia) cell forms [1]. Together with the cellular slime mold Dictyostelium discoideum and other Mycetozoa, P. polycephalum has been placed among the multicellular eukaryotes on the basis of molecular phylogenetic criteria [2]. The plasmodium undergoes mitosis without cytokinesis and usually develops into a multinuclear giant cell (macroplasmodium). This can contain billions of nuclei (e.g. 10 9 nuclei for a cell having a diameter of 14 cm), which divide synchronously within 3-4 min in an 8-9 h cycle. P. polycephalum, especially the plasmodium, has been chosen as a model to study biological and biochemical questions concerning differentiation and cell cycle [3,4].Considering the synchronous timing of cellular events and the giant dimension of a plasmodium, an appropriate device is necessary to achieve at any given moment an even distribution of molecular constituents. A characteristic oscillating protoplasma streaming [1] probably accounts for the travelling of material along the veins over a certain distance, although the mechanistic details are not known. To achieve a coordinated delivery of equilocally functioning proteins, molecular vehicles would have the advantage of stockpiling and carrying a number of different molecules jointly to defined loci, for example, nuclear p...

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confidence: 99%

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Karl¹,

Gasselmaier²,

Krieg

et al. 2003

European Journal of Biochemistry

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“…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 α and ε[13], and DNA polymerase δ[14] were consistent with the recovery of > 95% of DNA polymerase α and > 75% of the other DNA polymerases in the extract.…”

Section: Methodsmentioning

confidence: 78%

“…Nuclear extracts were prepared by incubating for 10 min on ice in an equal volume of extraction buffer (final concentrations 50 mM Tris/HCl pH 7.5, 0.3 M KCl, 20 mM MgCl 2 , 0.5% Triton X-100, 20% glycerol, 1 mM 2-mercaptoethanol, 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.…”

Section: Preparation Of Nuclear Extractmentioning

confidence: 73%

“…DNase‐Ι‐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 α and type ε, was prepared with a mixture of the purified P. polycephalum DNA polymerases [13], and rabbit antiserum against DNA polymerase δ by immunization with synthetic peptides of the enzyme [6]. Peroxidase‐coupled anti‐(rabbit IgG) Ig was purchased from Pierce.…”

Section: Methodsmentioning

confidence: 99%

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The DNA‐polymerase inhibiting activity of poly(β‐l‐malic acid) in nuclear extract during the cell cycle of Physarum polycephalum

Doerhoefer

Windisch

Angerer

et al. 2002

European Journal of Biochemistry

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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...

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“…To gain insight into evolutionary variability and synchrony maintenance, we began to characterize the replication apparatus of P. poly- cephalum. DNA polymerase α-primase complex α(140 kDa polymerizing subunit, two primase subunits of 53 and 58 kDa, and a 82 kDa polypeptide) (Weber et al, 1988; Achhammer etal., 1992; ), DNA polymerase δ(125 kDa) and PCNA (35 kDa) (Achhammer et al, 1995) have been identified and characterized. The DNA polymerase α 140 kDa subunit was proteolytically unstable, forming an inactive 110 kDa fragment (Weber et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling

et al. 1998

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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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DNA polymerase α-primase complex of Physarum polycephalum

Cited by 7 publications

References 10 publications

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

The DNA‐polymerase inhibiting activity of poly(β‐l‐malic acid) in nuclear extract during the cell cycle of Physarum polycephalum

Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling

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