Purification and properties of a pea chloroplast DNA polymerase

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.The chloroplast genome in higher plants exists as circular DNA molecules of 130-150 kilobase pairs (kbp). The chloroplast DNAs (ctDNAs) from higher plants have been found to be largely homogeneous in sequence organization as shown by renaturation kinetics, electron microscopy, denaturation mapping, and restriction endonuclease digestions (1-3). The replication of ctDNA has been studied by analyzing the replicating ctDNA molecules with electron microscopy (4,5

DNA polymerase activity of tomato fruit chromoplasts

“…Ethanol and cthidium bromide were also strong inhibitors, Aphidicolin, on the other hand, had no effect on the enzyme activity, and ddTTP, when used at a 1: 1 molar ratio with respect to dTTP, was a rather poor inhibitor (Table II). The previous results agree well with the general properties reported for plastid-type DNA polymerases [ 14,16,17].…”

“…The polymerases from both sources showed broad pH optima (between pH 7.5 and 8.5, Fig. 1A) in good agreement with previous reports [14]. Under standard crJnditions (pH 7.6) the maximal activity was obtained at 30'S (Fig.…”

“…In both cases the relative molecular mass was 88 template availability in chromoplasts, since relative kDA (Fig. 2B), which is in the range of previously reported values [ 14,16,17].…”

“…Chloroplast DNA polymerases are very minor peptides in leaf tissue [14,17], which has precluded so far Fruit maturation involves, among several other the preparation of specific antibodies. In the absence of changes, the conversion of photosynthetic chloroplasts into non-photosynthetic carotene-rich chromoplasts, a immunological methods of detection, we had to rely on activity measurements to estimate polymerase levels.…”

“…The other nuclear activity, chromatin-bound P-like polymerase, is highly resistant to NISI and araCTP [27j, whereas plastid enzymes were drastically inhibited (Table II). Our plastid preparations were substantially free from mitochondrial contamination (section 2) and their polymerase ac- [14,16,17], ineluding the apparent molecular mass of 88 kDa (Fig. 2).…”