Metal-dependent activity of I-PfoP3I.

<p>Plasmid DNA of pET<i>P3DNAP[−int]</i> (A) and PCR product of intron-minus version of Pf-WMP3 <i>DNAP</i> gene (B, C, D) were used as substrates. U, uncut substrates; L, BamHI-linearized plasmids; R (reference), substrates DNA digested by the metal-containing I-PfoP3I (without EDTA treated) with no externally added divalent ion; M, marker; S, substrate; P, product. Reactions were carried out at a fixed concentration (10 nM) of DNA substrates and (1000 nM) of apo-I-PfoP3I and metal-containing I-PfoP3I. (A, B) The concentrations of Mg²⁺ were 0, 1 pM, 1 nM, 1 µM, 1 mM, 10 mM and 50 mM. C. The effect of various metal ions on the nuclease activity of apo-I-PfoP3I. The reactions were carried out at a fixed concentration (10 mM) of different metal ions Mg²⁺, Ca²⁺, Mn²⁺ or without any metal ion (−Me²⁺). D. Different concentrations of Mg²⁺ (0, 50, 75, 100, 125 mM) were added to metal-containing I-PfoP3I. Lower concentration of Mg²⁺ had no effect on the nuclease activity of I-PfoP3I but over 100 mM Mg²⁺ of inhibited the enzyme activity. Ethidium bromide-stained agarose gels (1% agarose [wt vol⁻¹]) (A) or silver stained 6% urea acrylamide gels are shown (B, C, D).