Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site

Abstract

I-TevI, the T4 td intron-encoded endonuclease, catalyzes the first step in intron homing by making a double-strand break in the intronless allele within a sequence designated the homing site. The 28 kDa enzyme, which interacts with the homing site over a span of 37 bp, binds as a monomer, contacting two domains of the substrate. In this study, limited proteolysis experiments indicate that I-TevI consists of two domains that behave as discrete physical entities as judged by a number of functional and structural criteria. Overexpression clones for each domain were constructed and the proteins were purified. The carboxy-terminal domain has DNA-binding activity coincident with the primary binding region of the homing site and binds with the same affinity as the full-length enzyme. The isolated amino-terminal domain, contains the conserved GIY-YIG motif, consistent with its being the catalytic domain. Furthermore, site-directed mutagenesis of a conserved arginine residue within the extended motif rendered the full-length protein catalytically inactive, although DNA-binding was maintained. This is the first evidence that the GIY-YIG motif is important for catalytic activity. An enzyme with an N-terminal catalytic domain and a C-terminal DNA-binding domain connected by a flexible linker is in accord with the bipartite structure of the homing site.