The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from <i>Physarum polycephalum</i>

Zänker, Kurt S.; Schiebel, Winfried

doi:10.1042/bj1710445

Cited by 4 publications

(1 citation statement)

References 20 publications

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“…The form described by the sedimentation coefficient 5.5 S [3] and as form DE-111 enzyme [5] is probably identical with the HS2 DNA-synthesizing polypeptide, and their form C enzyme [2] with the HS1 DNA-polymerizing polypeptide. The results of the present investigation confirm and extend many of the previous findings.…”

Section: Discussionmentioning

confidence: 99%

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Holler

Fischer

Weber

et al. 1987

European Journal of Biochemistry

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Two forms of a DNA polymerase have been purified from microplasmodia of Physarurn polycephalurn by poly(ethy1eneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase CI 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 HSI 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 p 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.DNA polymerases of Physarurn polycephalum are of interest because (a) this organism is phylogenetically on the border between eucaryotes and procaryotes, and could therefore contain novel forms of DNA polymerases, and (b) Physarum macroplasmodia execute a naturally synchronous nuclear division (of more than lo8 nuclei/cell) making it an ideal model for cell cycle investigations [l].Recent studies [2 -51, however, have revealed considerable difficulties in obtaining and characterizing these DNA polymerases, probably due to interfering proteolysis and uncontrollabe factors in growing Physarurn microplasmodia. We have reinvestigated purification, during which we have monitored types of DNA polymerases as well as their molecular masses, and have used a newly developed combination of gradient gel electrophoresis with a DNA polymerase overlay assay (61. The method is less disruptive of weak complexes than, for instance, gel permeation chromatography. We report the characterization of a DNA polymerase which does not resemble any of those of higher eucaryotes. Correspondence to

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Section: Discussionmentioning

confidence: 99%

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Holler

Fischer

Weber

et al. 1987

European Journal of Biochemistry

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Organization and Replication of DNA in Physarum polycephalum

Evans¹

1982

Cell Biology of Physarum and Didymium

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Purification and caracterization of DNA polymerase α from plasmodia of Physarum polycephalum

Weber

Fischer

Holler

1988

European Journal of Biochemistry

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DNA polymerase a from Physarum polycephalum has been purified from freshly harvested microplasmodia. An inhibitory activity was removed by precipitation with poly(ethy1eneimine) and interfering type-/%like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases.The DNA-polymerizing polypeptide had a molecular mass of 140 kDa by polyacrylamide gel electrophoresis under denaturing conditions. It was contained in purified samples and in crude cell extracts. Peptides of smaller size that reacted with antibodies against this protein were generated during purification and prolonged standing. Molecular sizing under non-denaturing conditions resulted in high-molecular-mass forms.The type of isolated DNA polymerase was established on the basis of inhibition and template-primer utilization experiments underlying the classification of DNA polymerases from higher eucaryotes. The majority of the DNApolymerizing activity was contained in the cell nucleus fraction and was inhibited by aphidicolin.The isoelectric point (PI) was 6.7 f 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40°C. Monovalent salts, Li', Na', N H f , K', were inhibitory except for small activation maxima at 10 mM, 75 mM and 100 mM in the case of Na', NH: and K' respectively. The bivalent cations Mg" and Mn2* had broad activity maxima at 3-20 mM concentrations, which were shifted to 0.05-0.1 mM in the case of MnZ+ and synthetic DNA homopolymers.The numbers of molecules of DNA polymerases in Physarum nuclei were calculated and compared with the established number of replicons in plasmodia and with the number of molecules of DNA polymerases in higher eucaryotes.The purification of DNA polymerases from Physarum polycephalurn has been a standing issue since more than ten years [l -51. Despite a strong interest for synchronous nuclei division in the giant macroplasmodial cells of this organism [6], attempts to purify and characterize these enzymes have not been very successul. The reasons were several unusual properties of one of the DNA polymerases [7] and their instability, which probably exceeds that of most DNA Correspondence to

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The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum

Cited by 4 publications

References 20 publications

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Organization and Replication of DNA in Physarum polycephalum

Purification and caracterization of DNA polymerase α from plasmodia of Physarum polycephalum

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