Send to

Choose Destination
J Mol Biol. 1997 Feb 7;265(5):494-506.

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

Author information

Molecular Genetics Program, Wadsworth Center, New York State Department of Health and School of Public Health, State University of New York, Albany 12201-2002, USA.


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.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center