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An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase alpha in vitro
H Fischer1, S Erdmann, E Holler
1Institut für Biophysik und physikalische Biochemie, Universität Regenburg, West Germany.
Abstract:
From extracts of microplasmodia of Physarum polycephalum and their culture medium, an unusual substance was isolated which inhibited homologous DNA polymerase alpha of this slime mold but not beta-like DNA polymerase and not heterologous DNA polymerases. Analysis, especially NMR spectroscopy, revealed the major component to be an anionic polyester of L-malic acid and the inhibition to be due to poly(L-malate) in binding reversibly to DNA polymerase alpha. The mode of inhibition is competitive with substrate DNA and follows an inhibition constant Ki = 10 ng/mL. Inhibition is reversed in the presence of spermine, spermidine, poly(ethylene imine), and calf thymus histone H1. According to its ester nature, the inhibitor is slightly labile at neutral and instable at acid and alkaline conditions. Its largest size corresponds to a molecular mass of 40-50 kDa, but the bulk of the material after purification has lower molecular masses. The inhibitory activity depends on the polymer size and has a minimal size requirement.
Insights
Researchers discovered poly(L-malate), a substance from slime mold, that inhibits DNA polymerase alpha. This finding offers new insights into enzyme inhibition and potential molecular targets.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Slime mold Physarum polycephalum produces unique biomolecules.
- DNA polymerase alpha is crucial for DNA replication and repair.
Purpose of the Study:
- To isolate and characterize an inhibitor of Physarum polycephalum DNA polymerase alpha.
- To elucidate the mechanism of inhibition and the chemical nature of the inhibitor.
Main Methods:
- Extraction and purification of the inhibitory substance from Physarum polycephalum.
- Biochemical assays to determine enzyme inhibition kinetics.
- Nuclear Magnetic Resonance (NMR) spectroscopy for structural elucidation.
Main Results:
- An anionic polyester of L-malic acid, identified as poly(L-malate), was isolated.
- Poly(L-malate) reversibly inhibits Physarum polycephalum DNA polymerase alpha competitively with substrate DNA (Ki = 10 ng/mL).
- Inhibition is dependent on polymer size and can be reversed by polyamines and histone H1.
Conclusions:
- Poly(L-malate) is a novel, size-dependent inhibitor of DNA polymerase alpha.
- The discovery provides a new tool for studying DNA polymerase function.
- This research highlights the potential of microbial natural products in enzyme inhibition studies.