Structural and functional organization of I-TevI, a homing endonuclease of the GIY-YIG family

The thymidylate synthase gene of bacteriophage T4 is divided into two exons by the presence of a self-splicing group I intron. Encoded by an open reading frame within the intron is a homing endonuclease of the GIY-YIG family, I-TevI. The td intron is a mobile intron. I-TevI recognizes and cleaves intronless td alleles and repair of the double-strand break using an intron-plus td allele results in conversion of the intronless allele to intron-plus.; I-TevI is structurally and functionally divided into two domains by a flexible tether; an N-terminal catalytic domain and a C-terminal DNA-binding domain. The N-terminal domain contains the GIY-YIG conserved sequence module common among the other members of the GIY-YIG family. I-Tev I is a sequence specific enzyme but, unlike the restriction enzymes, it is also tolerant of sequence polymorphisms, consistent with theories about the evolution of homing endonucleases. This tolerance makes the enzyme difficult to work with since overexpression of quantities sufficient for physical analyses is toxic to E. coli, presumably due to non-specific cutting of the host chromosome. In order to study I-TevI structurally, the two domains have been separated and their structures solved separately. The C-terminal domain by itself is nontoxic and its structure, bound to DNA, has been solved by X-ray crystallography. The crystal structure reveals a unique wrap-around structure. The N-terminal domain with wild type sequence is to toxic to overexpress; so catalytic mutants (R27A and E75A) were overexpressed and studied by both NMR spectroscopy and X-ray crystallography.; Studies of the N-terminal domain by NMR reveal a ββααβα secondary structure and those residues beyond A92, which is the last residue of the GIY-YIG module, are unstructured. Solution of the N-terminal domain crystal structure reveals a globular domain with the expected secondary structure elements. The putative catalytic site, identified by the location of residues R27 and E75, is on a shallow, concave surface c the domain. The N-terminal domain was titrated with Mn2+ and a metal binding site identified by chemical shift mapping. The site is likely a non-specific binding site since does not involve any of the most conserved residues of the GIY-YIG module. Thus, despite the structures now available for I- TevI, mechanistic data is still lacking.