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Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions
E Holler1, G Achhammer, B Angerer
1Institut für Biophysik und physikalische Biochemie der Universität Regensburg, Federal Republic of Germany.
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-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-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(vinyl 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 (N) 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(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-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-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.
Insights
Poly(L-malate), a nuclear polyanion in Physarum polycephalum, inhibits DNA polymerase activity and binds histones. Its interaction with DNA polymerase depends on charge spacing, suggesting roles in cell cycle regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Poly(L-malate) is an unusual polyanion identified in the nuclei of Physarum polycephalum plasmodia.
- Understanding its interactions with key cellular machinery like DNA polymerase and histones is crucial for elucidating its biological functions.
Purpose of the Study:
- To investigate the interaction of poly(L-malate) and related polyanions with the DNA-polymerase-alpha-primase complex and histones from P. polycephalum.
- To determine the structure-activity relationship of polyanions in inhibiting DNA polymerase activity.
Main Methods:
- Enzymatic assays were employed to measure DNA polymerase activity.
- Fluorimetric methods were used to assess binding interactions.
- A range of polyanions with varying charge spacing were tested for their inhibitory potency.
Main Results:
- Poly(L-malate) was found to inhibit the DNA-polymerase-alpha-primase complex and bind to histones.
- Inhibition of DNA polymerase activity by polyanions correlated with the distance between neighboring charges.
- Poly(L-malate) and poly(D,L-malate) exhibited the highest inhibitory potency, with N=5 (distance between charges).
- Spermidine, spermine, and histones antagonized the inhibitory effects of poly(L-malate).
Conclusions:
- Poly(L-malate) likely interacts with the DNA-polymerase-alpha-primase complex in P. polycephalum.
- Poly(L-malate) may function as a molecular chaperone during nucleosome assembly in the S phase.
- It may also act as an inhibitor and storage agent for DNA-polymerase-alpha-primase during the G2 and M phases of the cell cycle.