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

Abstract

The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase E and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-l inked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The a, 6, E and /?-like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by /?-poly(~-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase E and a 135 kDa /?-like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei a t growth arrest.

The DNA‐polymerase inhibiting activity of poly(β‐l‐malic acid) in nuclear extract during the cell cycle of Physarum polycephalum

“…In previous analytical and preparative experiments, it has been shown that PMLA was the constituent that specifically inhibited replicative DNA polymerases in nuclear extracts [1,2,7]. In the present study, we measured the inhibitory activity of PMLA using purified DNA polymerase α of P. polycephalum as an added reporter.…”

“…Labeling of DNA polymerases was carried out in a 20‐µL solution containing 5 µL nuclear extract, 3–5 µ m [ 32 P]dATP (3000 Ci·mmol −1 ), 125 µ m AFBdCTP, 33 µ m of each dGTP and dTTP, 50 m m 3‐( N ‐morpholino)propanesulfonic acid buffer (pH 7.5), 50 m m KCl, 10 m m MgCl 2 , 3 m m EDTA and 5 µg activated salmon testis DNA [7]. Escherichia coli DNA polymerase I served as a positive control in a parallel, but otherwise identical, reaction mixture.…”

Photoreactive DNA Probes as a Tool for Studying the Translesion Synthesis System in Mammalian Cell Extracts

“…Such approach was successfully applied for investigation of the relationships among DNA polymerases and auxiliary proteins as well as DNA-interaction proteins and DNA in reconstituted systems [12][13][14][15][16] and in nuclear/cellular extracts [17][18][19]. The TLS-system is very complex and dynamic to be studied by X-ray analysis or other instrumental techniques.…”

“…Several of such dNTP analogues were shown to be substrates of viral, prokaryotic and eukaryotic DNA polymerases [17,[21][22][23][24][25][26][27] and potentially can be introduced by TLS polymerases. Photochemical properties of the photoreactive groups have been adjusted for using excitation near UV light to avoid stimulation of the intrinsic photoreactivity of the nucleic acids and proteins.…”

A Binary System of Photoreagents for High-Efficiency Labeling of DNA Polymerases

“…It creates serious problems at the stage of purification and sequencing the crosslinked peptides by the Edman procedure or by MALDI mass-spectroscopy. Low crosslinking efficiency of arylazido derivatives of nucleic acids results in poor identification of DNA polymerases or accompanied proteins in the crude cellular or nuclear extracts (6). Therefore future application of photoaffinity labeling technique to study replication machinery demands improved efficiency and specificity of photoaffinity labeling of DNA polymerases.…”