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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Apr;81(8):2354–2358. doi: 10.1073/pnas.81.8.2354

Purification and properties of a pea chloroplast DNA polymerase

R L McKown 1, K K Tewari 1
PMCID: PMC345058  PMID: 16593454

Abstract

A DNA polymerase has been purified >3,000-fold from the chloroplasts of pea plants by chromatography on DEAE-cellulose, phosphocellulose, single-stranded DNA-agarose, and sedimentation in a glycerol gradient. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate indicates that the final fraction contained a single discernible protein band of 90,000 daltons. Gel filtration on Sephacryl S-200 and glycerol gradient sedimentation under nondenaturing conditions demonstrate that the chloroplast DNA polymerase has a native molecular mass of approximately 87,000 daltons. The purified polymerase lacks any associated nuclease activity. The enzyme activity is inhibited by N-ethylmaleimide (74% at 1.0 mM) and ethidium bromide (90% at 0.23 mM) and is resistant to aphidicolin. The purified enzyme is totally dependent on the presence of added DNA, has an absolute requirement for Mg2+ (12 mM optimal), is stimulated by K+ (120 mM optimal), and requires all four deoxynucleoside triphosphates for maximum activity. Native DNA which has been degraded to a limited extent with DNase I is the most efficient template.

Keywords: circular DNA, DNA replication

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Selected References

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