FIGURE 6.

Divalent ion requirements for optimal CPV resolvase activity. A–C, Mg²⁺ alone as cofactor (A), Mn²⁺ alone as cofactor (B), and Mn²⁺ fixed at four different concentrations (C), with Mg²⁺ varied for each. D, Mg²⁺ and Mn²⁺ required for HJ cleavage by the CPV resolvase D130N mutant. The D130N substitution weakens A site metal binding, requiring ∼10-fold higher concentration of either metal ion. The assay in A–D monitors cleavage of 10 nm fluorescently labeled HJ substrate by 100 nm resolvase after 30 min. The arrows indicate the positions of substrate (S) and product (P) bands on native PAGE after quenching the reactions.