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

Abstract

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

Specific inhibition of Physarum polycephalum DNA‐polymerase‐α‐primase by poly(l‐malate) and related polyanions

“…The purification of DNA polymerase a and of p-like DNA polymerase has been described before [8,91. Unless purified over heparinSepharose, preparations contained DNA primase firmly bound to DNA polymerase a. Primase from P. polycephalum was a heterodimer of proteins with molecular masses 52 kDa and 57 kDa, as judged by SDS gel electrophoresis after autocatalytic labelling with either the 4-hydroxybenzaldehyde ester or the 2-hydroxybenzaldehyde ester of ATP according to [ 4°C.…”

Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling

“…(1988) and Achhammer et al (1992). P. polycephalum DNA polymerase S was purified as described by Achhammer et al (1995). The enzymes were stored at -70 "C in the presence of 50% (v/v) glycerol, 50 mM Tris/HCl pH 7.5, 1 mM EDTA and 1 mM 2-mercaptoethanol.…”

“…The primer was 5'-end labelled with [Y-~~PIATP as described by Sambrook et al (1989), and was separated from excess reactants on NENSORB-20 (DuPont). Other synthetic template-primers were poly (rA)/p(dT),,-,, (lo/ 1), poly (dA)/ according to Achhammer et al (1995). DNA polymerases.…”

“…To gain insight into evolutionary variability and synchrony maintenance, we began to characterize the replication apparatus of P. polycephafum. DNA polymerase u-primase complex (140 kDa polymerizing subunit, two primase subunits of 53 and 58 kDa, and a 82 kDa polypeptide) (Weber et al, 1988;Achhammer et al, 1992;, DNA polymerase 6 (125 kDa) and PCNA (35 kDa) (Achhammer et al, 1995) have been identified and characterized. The DNA polymerase a 140 kDa subunit was proteolytically unstable, forming an inactive 110 kDa fragment (Weber et al, 1988).…”

“…DNA polymerase u-primase complex (140 kDa polymerizing subunit, two primase subunits of 53 and 58 kDa, and a 82 kDa polypeptide) (Weber et al, 1988;Achhammer et al, 1992;, DNA polymerase 6 (125 kDa) and PCNA (35 kDa) (Achhammer et al, 1995) have been identified and characterized. The DNA polymerase a 140 kDa subunit was proteolytically unstable, forming an inactive 110 kDa fragment (Weber et al, 1988). The polymerase was found to differ in several respects from polymerases of yeasts and higher eukaryotes (Achhammer et al, 1995).…”