DNA-BINDING AND CLEAVAGE BY THE NUCLEAR INTRON-ENCODED HOMING ENDONUCLEASE I-PPOI

Homing endonucleases are a diverse collection of proteins that are enc oded by genes with mobile, self-splicing introns(1-3). They have also been identified in self-splicing inteins (protein introns)(4). These e nzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-spe cific double-strand break at a target site in an allele that lacks the corresponding mobile intron(3). The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four famili es of homing endonucleases have been identified, including the LAGLIDA DG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases(1). The f irst identified His-Cys box homing endonuclease was I-PpoI from the sl ime mould Physarum polycephalum(5,6). Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility(7). Here we re port the structure of the I-PpoI homing endonuclease bound to homing-s ite DNA determined to 1.8 Angstrom resolution. I-PpoI displays an elon gated fold of dimensions 25 x 35 x 80 Angstrom, with mixed (alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long ol-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 Angstrom apart. The enzyme pos sesses a new zinc-bound fold and endonuclease active site. The structu re has been determined in both uncleaved substrate and cleaved product complexes.