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Experimental Mycology

R.C. Staples

A DNA-dependent DNA polymerase was extracted from resting uredospores of the bean rust fungus and purified. The enzyme was readily soluble when extracted from resting spores and was purified 300-fold from the crude spore extract. While not retained on phosphocellulose, the enzyme was eluted as a single peak from columns of DEAE-cellulose, DNA-cellulose, or polyacrylamide gels. Its molecular weight was estimated to be about 175,000. The activity of DNA polymerase depended upon the simultaneous presence of dATP, dCTP, dTTP, dGTP, a DNA template, and magnesium ions. Optimum pH of the reaction was 7.5. Activated DNA was preferred by the enzyme as a template. The product synthesized in a cell-free system was solubilized by treatment with deoxyribonuclease and had the same buoyant density as the DNA used for the template. The purified protein from resting spores was resolved by isolelectric focusing on density gradients of glycerol into four active fractions. Two of these, at pH 5.4 and 7.0, were relatively abundant, while at least two minor fractions were present at pH 6.3 and 8.3. All enzymes were inhibited by N-ethylmaleimide (NEM). The pI 5.4 enzyme was especially active with poly(dA)(dT)10 as primer.

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DNA polymerase from nongerminated bean rust uredospores
2

R.C. Staples

DNA polymerase from nongerminated bean rust uredospores

A DNA-dependent DNA polymerase was extracted from resting uredospores of the bean rust fungus and purified. The enzyme was readily soluble when extracted from resting spores and was purified 300-fold from the crude spore extract. While not retained on phosphocellulose, the enzyme was eluted as a single peak from columns of DEAE-cellulose, DNA-cellulose, or polyacrylamide gels. Its molecular weight was estimated to be about 175,000. The activity of DNA polymerase depended upon the simultaneous presence of dATP, dCTP, dTTP, dGTP, a DNA template, and magnesium ions. Optimum pH of the reaction was 7.5. Activated DNA was preferred by the enzyme as a template. The product synthesized in a cell-free system was solubilized by treatment with deoxyribonuclease and had the same buoyant density as the DNA used for the template. The purified protein from resting spores was resolved by isolelectric focusing on density gradients of glycerol into four active fractions. Two of these, at pH 5.4 and 7.0, were relatively abundant, while at least two minor fractions were present at pH 6.3 and 8.3. All enzymes were inhibited by N-ethylmaleimide (NEM). The pI 5.4 enzyme was especially active with poly(dA)(dT)10 as primer.

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