Specific inhibition of Physarum polycephalum DNA‐polymerase‐α‐primase by poly(l‐malate) and related polyanions

Abstract

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 [...

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“…Structurally, poly(α,β-(D,L)-aspartic acid) and poly(α,β-(D,L)-malic acid) are similar except the replacement of the amido group in PAA by the ester group in PMLA. Remarkably, the spatial distances of polymer pendant carboxylates are similar, which was recognized in experiments measuring the inhibition of DNA polymerases by polyanions of variable structures [44]. In this study, a similarity of the distance between the negatively charged carboxylates in PMLA and PAA with phosphate groups in nucleic acids has been noted.…”

“…Polymalic acid of high molecular weight was discovered by its ability to mimic nucleic acids and competitively inhibit DNA polymerase α of Physarum polycephalum , and, to a lesser extent, the activities of DNA polymerases from other organisms [43, 44] (Fig. 3).…”

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly

“…Subsequent work showed that the slow addition of histone made it possible to assemble nucleosomes without the histone chaperone at near physiological ionic strength. Under these conditions, however, nucleosome assembly could be stimulated by the addition of other chromatin components (e.g., the linker histone H1 and the non-histone protein HMG [7,8]) and acidic polymers (e.g., acidic polypeptide [9,10], poly (L-malate) [11], and RNA [12]). These studies resulted in the isolation of other histone chaperones with different primary structures and demonstrated that there are various types of histone chaperones [13].…”

Preparation of Free and of Specifically Protected Oligo[β-Malic Acids] for Enzymatic Degradation Studies

“…for DNA polymerases) and is therefore assumed to be involved in DNA replication. [17] The organisms most efficient in producing β-PMA are the black yeasts Aureobasidium sp. [18] and A. pullulans, [19] from which up to 61 g/l and 9.8 g/l culture medium were isolated, respectively.…”