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A DNA polymerase with unusual properties from the slime mold Physarum polycephalum
Abstract:
Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase alpha on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzyme was 30-40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase beta by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.
Insights
Researchers purified two DNA polymerase forms from Physarum polycephalum. These enzymes resemble eukaryotic DNA polymerase beta and may function as bacterial-like repair DNA polymerases in lower eukaryotes.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- DNA polymerases are crucial for DNA replication and repair.
- Eukaryotic DNA polymerases exhibit diverse structures and functions.
- Lower eukaryotes may possess unique DNA polymerase systems.
Purpose of the Study:
- To purify and characterize DNA polymerases from Physarum polycephalum.
- To investigate the relationship between different DNA polymerase forms.
- To compare Physarum DNA polymerases with known eukaryotic and prokaryotic enzymes.
Main Methods:
- Purification using poly(ethyleneimine) precipitation and various chromatography techniques (DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose).
- Separation of DNA polymerase alpha and distinct HS1 and HS2 forms.
- Analysis of enzyme composition and stoichiometry using protein complexes.
Main Results:
- Two distinct DNA polymerase forms (HS1 and HS2) were purified from Physarum polycephalum.
- Form HS2 was derived from form HS1, suggesting a relationship possibly involving proteolysis.
- Both forms consisted of a 60-kDa protein associated with a DNA-polymerizing polypeptide (135-kDa for HS1, 110-kDa for HS2) in a 1:1 molar ratio.
- The purified DNA polymerases showed similarities to eukaryotic DNA polymerase beta and were functionally indistinguishable.
Conclusions:
- Physarum polycephalum possesses at least two forms of DNA polymerase.
- These enzymes are structurally and functionally similar to eukaryotic DNA polymerase beta.
- It is proposed that lower eukaryotes harbor repair DNA polymerases analogous to bacterial enzymes on a molecular mass basis.