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 [...
DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.
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.
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