PROTEIN FOOTPRINTING APPROACH TO MAPPING DNA-BINDING SITES OF 2 ARCHAEAL HOMING ENZYMES - EVIDENCE FOR A 2-DOMAIN PROTEIN-STRUCTURE

The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron(-) or intein (-) alleles site-specifically, and thereby facilitate homing of the in trons or inteins which encode them. The protein structure and the mech anism of DNA recognition of these homing enzymes is largely unknown. T herefore, we examined these properties of I-Pod and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage w ithin regions that are equidistant from each of the two LAGLIDADG moti fs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 a mino acids after each of the LAGLIDADG motifs, were protected, Our dat a suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DN A binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consisten t with a model where the LAGLIDADG endonucleases bind to their non-pal indrommic substrates as monomeric enzymes, with each of the two domain s recognizing one half of the DNA substrate.