Generation of Highly Site-Specific DNA Double-Strand Breaks in Human Cells by the Homing Endonucleases I-PpoI and I-CreI

Abstract

We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycetePhysarum polycephalumand I-CreI from the green algaChlamydomonas reinhardtii,to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3′ ends that initiate target intron transposition or “homing.” We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repairedin vivo,although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.