Purification and properties of a DNA primase from Nicotiana tabacum.
A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase alpha-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase.
Garcia-Maya
M M
MM
Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
Buck
K W
KW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D019915:DNA Primase / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010947:Plants, Toxic; D012293:Rifampin / Q000494:pharmacology; D014026:Nicotiana / Q000201:enzymology*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D016590:Aphidicolin; D000255:Adenosine Triphosphate; D019915:DNA Primase; D008274:Magnesium; D005033:Ethylmaleimide; D012293:Rifampin