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A meiotic DNA polymerase from a mushroom, Agaricus bisporus
1Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Chiba-ken, Japan.
Abstract:
A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.
Insights
Researchers isolated a meiotic DNA polymerase from Agaricus bisporus mushrooms, likely involved in DNA repair. This beta-like enzyme shows optimal activity under meiotic conditions and cross-reacts with antibodies against a related fungal polymerase.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- DNA polymerases are crucial for DNA replication and repair.
- Meiosis involves specialized DNA replication and repair processes.
- Fungal DNA polymerases offer insights into conserved biological mechanisms.
Purpose of the Study:
- To isolate and characterize a meiotic DNA polymerase from Agaricus bisporus.
- To investigate the enzyme's potential role in meiotic DNA repair.
- To compare its properties with known fungal DNA polymerases.
Main Methods:
- Purification of DNA polymerase from Agaricus bisporus.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular mass determination.
- Enzyme activity assays under varying pH, ionic strength, and temperature conditions.
- N-ethylmaleimide and 2',3'-dideoxythymidine 5'-triphosphate sensitivity tests.
- Western blot analysis using antiserum against Coprinus DNA polymerase beta.
Main Results:
- A meiotic DNA polymerase fraction was purified, showing three protein bands (72 kDa, 65 kDa, 36 kDa).
- Optimal activity was observed at pH 7.0-8.0, 5 mM Mg2+, 50 mM KCl, and 28-30°C.
- The enzyme exhibited characteristics of a beta-like DNA polymerase (N-ethylmaleimide resistant, ddTTP sensitive).
- Western blot analysis confirmed cross-reactivity with antiserum against Coprinus DNA polymerase beta, specifically with the 65 kDa band.
- The enzyme's activity was primarily found in meiotic cells, though present in an inactive state in somatic cells.
- Antiserum cross-reacted with extracts from various mushroom families and Saccharomyces.
Conclusions:
- An active meiotic DNA polymerase was identified in Agaricus bisporus, likely involved in meiotic DNA repair.
- The enzyme shares characteristics with beta-like DNA polymerases and shows evolutionary conservation across fungal species.
- Further research can elucidate the specific role of this polymerase in meiotic processes.