PROFILE OF THE DNA RECOGNITION SITE OF THE ARCHAEAL HOMING ENDONUCLEASE I-DMOI

I-DmoI is a homing enzyme of the LAGLI-DADG type that recognizes up to 20 bp of DNA and is encoded by an archaeal intron of the hyperthermop hilic archaeon Desulfurococcus mobilis. A combined mutational and DNA footprinting approach was employed to investigate the specificity of t he I-DmoI-substrate interaction. The results indicate that the enzyme binds primarily to short base paired regions that border the sites of DNA cleavage and intron insertion. The minimal substrate spans no more than 15 bp and while sequence degeneracy is tolerated in the DNA bind ing regions, the sequence and size of the cleavage region is highly co nserved. The enzyme has a slow turnover rate and cuts the coding stran d with a slight preference over the non-coding strand. Complex formati on produces some distortion of the DNA double helix within the cleavag e region. The data are compatible with the two DNA-binding domains of I-DmoI bridging the minor groove, where cleavage occurs, and interacti ng within the major groove on either side, thereby stabilizing a disto rted DNA double helix. This may provide a general mode of DNA interact ion at least for the LAGLIDADG-type homing enzymes.