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Abstract
NaeI endonuclease has been studied as a model system for understanding DNA recognition by proteins because of its elaborate interactions with DNA. NaeI is a member of a subclass of type II restriction enzymes that require the interaction of two sequences in order for cleavage to occur. NaeI has two DNA-binding sites, one binds the enhancer DNA which stimulates cleavage, and the other binds the substrate DNA which is cleaved. Both the enhancer and substrate DNAs contain the recognition site GCCGGC, but differ in flanking sequences.
In cleavage assays, a transition was observed from single-strand to double-strand cleavage activity, indicating a quaternary change in the enzyme. Gel mobility-shift assay of the interaction of NaeI with enhancer showed monomer and dimer complexes. Dimer formation increased with the affinity of enhancer for NaeI. These results imply a model for cleavage enhancement in which enhancer-binding stabilizes an active NaeI dimer conformation.
The monomeric structure of NaeI was stabilized by the addition of glycerol, DMSO, and Mn$\sp{2+}$. The NaeI activity was thus converted from double-strand to single-strand scission of DNA. Rates of first- and second-strand nicking demonstrated that NaeI monomer nicks the two DNA strands of a recognition site independently. Nicking the second strand was much slower than nicking the first strand because monomer has difficulty dissociating from fully cleaved product.
To further elucidate the NaeI-DNA interaction, the role of the divalent cation was examined. Ca$\sp{2+}$ substituted for Mg$\sp{2+}$ in the reactions gave very stable protein-DNA complexes and provided conditions in which dissociation constants and thermodynamic parameters could be accurately measured. Substitution for Mg$\sp{2+}$ by other divalent cations in NaeI reactions demonstrated one group that allowed tight binding of DNA without cleavage (Ca$\sp{2+}$, Ni$\sp{2+}$, Cu$\sp{2+}$) and one group that allowed the enzyme to proceed in cleavage (Mg$\sp{2+}$, Mn$\sp{2+}$, Fe$\sp{2+}$, Co$\sp{2+}$). These results suggest a dual role for the divalent in the NaeI reaction: (1) Binding, (2) Catalysis.
