Protein Engineering Alfred Pingoud, Ann-Josée Noël, Vera Pingoud, Shawn Steuer, and

Wolfgang Wende

Justus-Liebig-Universität Giessen, Germany


1.1Structure, Function, and Evolution of Homing Endonucleases

Homing endonucleases are a recently discovered class of enzymes (reviews: 1-5). To date (May 2002), 92 different homing endonucleases have been identified (https://rebase.neb.com/rebase/link_homing (6). They all recognize extended specific DNA sequences of up to 40 base pairs in length and cleave the DNA in both strands of the recognition sequence or nearby, thereby producing DNA fragments with sticky ends. These fragments are recombinogenic and, provided that homologous sequences are present, may lead to the integration of homing endonuclease genes by a double-strand break repair mechanism into alleles that lack them (Fig. 1). This process of unilateral transfer of an intervening sequence was called “homing” and the endonucleases that initiate this process were therefore called “homing endonucleases” (7, 8). The reaction is not reversible because the cleavage site for

Figure 1 The homing event. Homing is a site-specific unidirectional transfer of a mobile genetic element (an intein or a group I or II intron) from one allele to a homologous allele lacking this element. The process is initiated by a double-strand cut in the recipient allele, catalyzed by a homing endonuclease that is usually inteinencoded such as PI-SceI (left), or intron-encoded such as I-CreI (right). Double-strand break repair, which involves duplication of the mobile element in the donor allele, leads to the specific insertion of the mobile element coding for the homing endonuclease in the recipient allele by homologous recombination.