Homing Endonucleases Encoded by Germ Line-Limited Genes in Tetrahymena thermophila Have APETELA2 DNA Binding Domains

Three insertion elements were previously found in a family of germ line-limited mobile elements, the Tlr elements, in the ciliate Tetrahymena. Each of the insertions contains an open reading frame (ORF). Sequence analysis of the deduced proteins encoded by the elements suggests that they are homing endonucleases. The genes are designated TIE1-1, TIE2-1, and TIE3-1 for Tetrahymena insertion-homing endonuclease. The endonuclease motif occupies the amino terminal half of each TIE protein. The C-terminal regions of the proteins are similar to the APETELA2 DNA binding domain of plant transcription factors. The TIE1 and TIE3 elements belong to families of repeated sequences in the germ line micronuclear genome. Comparison of the genes and the deduced proteins they encode suggests that there are at least two distinct families of homing endonuclease genes, each of which appears to be preferentially associated with a specific region of the Tlr elements. The TIE1 and TIE3 elements and their cognates undergo programmed elimination from the developing somatic macronucleus of Tetrahymena. The possible role of homing endonuclease-like genes in the DNA breakage step in developmentally programmed DNA elimination in Tetrahymena is discussed. Homing endonucleases are encoded by insertion elements. These elements are most often introns or inteins but can also be freestanding elements (14). The endonucleases make a double- or single-stranded cut in the DNA of a homologous allele lacking the element. The break in the DNA initiates the transposition of the element into the empty allele, a process known as homing (5). Transposition of the element occurs via a gene conversion event that is completed by the DNA repair systems of the host organism. Homing endonucleases are site specific. Their recognition sites, at 12 to 40 bp, are much longer than those of most restriction enzymes. This limits the range of potential homing sites. The homing endonucleases are also distinct from restriction enzymes in that they can tolerate changes of a few base pairs in the recognition site. This provides flexibility such that the element can sometimes home into a site that has been slightly modified by mutation. Homing endonucleases have been classified into four groups, based on the structures of the catalytic domains (5). The most widespread and well studied of these enzymes have one or more copies of the LAGLIDADG motif. The structures of several of these enzymes, bound to their target sites, have been solved (9, 17, 20, 26). They bind as dimers or pseudodimers to their substrates, and in a few cases the catalytic mechanisms of DNA cleavage are understood in detail. The second group of homing endonucleases are characterized by the motif GIY(X10-11)YIG. These are monomeric enzymes that cleave the DNA at a distance from the intron insertion site. The third class, the His-Cys box enzymes, are characterized by conservation of histidine and cysteine residues over a stretch of 100 amino acids. The least understood class of enzymes are the HNH (or in some cases, HNN) endonucleases. The catalytic sites of these enzymes contain histidine and asparagine residues with characteristic spacing over a range of 30 to 33 residues.