Purification and properties of a DNA primase from Nicotianatabacum |
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Authors: | Mitla M Garcia-Maya Kenneth W Buck |
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Institution: | (1) Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK, GB |
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Abstract: | 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 α-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.
Received: 8 May 1997 / Accepted: 5 June 1997 |
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Keywords: | : DNA primase Nicotiana (DNA primase) Oligoribonucleotide primers |
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