The characteristics of spinach chloroplast DNA polymerase

Abstract

Purified chloroplast preparations from spinach incorporate [ 3H]-dTTP 1 1 Abbreviations: dTTP, dGTP, dATP and dCTP, the triphosphates of deoxythymidine, deoxyguanosine, deoxyadenosine and deoxycytidine respectively; DNA, deoxyribonucleic acid; Tris, tris(hydroxymethyl) aminomethane; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid; CAP, chloramphenicol; TCA, trichloracetic acid; SSC, 0.15 m NaCl plus 0.015 m Na-citrate. into an acid-insoluble product. The incorporation reaction is highly dependent on all four deoxynucleoside triphosphates, Mg 2+ and a sulphydryl reagent, and is further stimulated by KCl. The reaction is inhibited by deoxyribonuclease, actinomycin D and p-hydroxymercuribenzoate. Neither DNA nor the associated DNA polymerase activity is readily washed out of chloroplasts by hypotonic solutions. The DNA polymerase system of chloroplasts is more active than that of leaf nuclei both per unit DNA and per cell. The radioactive product of the incorporation reaction in chloroplasts sedimented at 18S in neutral sucrose density gradients and at 6–7S under alkaline conditions. The buoyant density of the radioactive chloroplast product increased upon denaturation and decreased upon subsequent renaturation, and was coincident with chloroplast DNA under all conditions. It did not coincide with nuclear DNA following denaturation and renaturation. DNA-DNA hybridization studies showed a considerably greater interaction between the radioactive chloroplast product and chloroplast DNA than with nuclear DNA. Chloroplasts also contain a soluble and readily leached DNA polymerase fraction which can be primed with exogenous plant DNAs.