Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI

Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are cla ssified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymme tric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 Angstrom crystal structure of the archaeal PI-PfuI in tein from Pyroccocus furiosus. The structure reveals a unique domain, desig nated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a ca talytic center for protein splicing, which involves both N and C-terminal r esidues. The endonuclease domain, which is inserted into the Hint domain, c onsists of two copies of substructure related by an internal pseudo 2-fold axis. In Contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-blnding cle ft formed by a pair of four-stranded beta-sheets. DNase I footprinting expe riments showed that PI-PfuI covers more than 30 bp of the substrate asymmet rically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompas sing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, t he two strands in the DNA duplex were cleaved by PI-PfuI with different eff iciencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites. (C) 2000 Academic Press.