The Recognition Sites of Free Standing Endonucleases Are Distinct from the Endonuclease Insertion Site

Phage-encoded free-standing endonucleases that have been characterized to date all introduce a DSB, leaving a 3' 2-nucleotide overhang (Belle et al. 2002; Liu et al. 2003), similar to GIY-YIG intron-encoded endonucleases (Bell-Ped-ersen et al. 1989; Loizos et al. 1996; Edgell and Shub 2001). While the biochemistry of DNA cleavage may be similar to intron-encoded versions, free-stand-

Fig. 3. Mechanisms to minimize cleavage of host genomes differ for a intron-encoded or b free-standing endonucleases. Black triangles represent endonuclease cleavage sites. X indicates a nucleotide substitution that may prevent cleavage by free-standing endonucleases

ing endonucleases differ from intron endonucleases in one key property, the separation of endonuclease cleavage and endonuclease gene insertion sites, which are often hundreds of base pairs distant (Fig. 3; Edgell 2002). This arrangement contrasts with that of intron-encoded endonucleases, where the endonuclease cleavage and intron insertion sites are separated by 2-25 base pairs (Chevalier and Stoddard 2001).

For intron- or intein-encoded endonucleases, the recognition site and cleavage sites are intimately linked to the insertion site of the genetic element. This is due to the fact that intron- or intein-endonuclease recognition sites encompass sequence up- and down-stream of the insertion site. Compilation of the insertion sites of self-splicing introns and inteins has revealed that many interrupt conserved protein-coding genes (Dalgaard et al. 1997; Edgell et al. 2000; Pietrokovski 2001; Gogarten et al. 2002). Furthermore, the intron or in-tein insertion site often lies within, or nearby, nucleotide sequence that corresponds to functionally critical residues of the host protein. Thus, the recognition site of an intron- or intein-encoded endonuclease will include the conserved nucleotide sequence(s) that surround the insertion site (Edgell et al. 2000, 2003). These nucleotide sequences are those that are likely to be conserved between related genomes, thus maximizing the potential of the endonuclease to spread to related genomes.

In theory, free-standing endonucleases could use the junction of the endonuclease insertion site as a recognition sequence, in analogy to the recognition sequences of intron- or intein-encoded endonucleases. Such sequences, however, are likely to be poor choices for a recognition site because of the potential for variability in intergenic regions of phage DNA. Mapping of the cleavage sites for SegE, SegF and SegG all demonstrated that they lie within genes adjacent to the Seg insertion site, and that the target genes are conserved between T-even phage genomes (Kadyrov et al. 1997; Belle et al. 2002; Liu et al. 2003). This strategy of selection of a recognition site within a conserved gene is similar to that employed by intron-encoded versions, as it maximizes the potential that the site will be present in related genomes.

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