Homing endonuclease I-CreII: a novel dual-motif enzyme that catalyzes group I intron homing

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2005

Authors

Corina, Laura Elizabeth

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Abstract

I-CreII is a homing endonuclease from the Cr.psbA intron of Chlamydomonas reinhardtii. It cleaves the exon 4-exon 5 junction of psbA DNA, thereby inducing a recombination event referred to as intron homing. Homing endonucleases are classified into four main groups based on the presence of a catalytic domain unique to each group. I-CreII is novel in that it appears to contain two catalytic domains, an H-N-H and a GIY-YIG. The role of the putative GIY-YIG motif has been questioned, however, because I-CmoeI, a related protein, behaves like an H-N-H endonuclease (Drouin et al., 2000). Recently, Kim et al. (2005) showed that DNA cleavage by I-CreII leaves 2-nt 3′ OH overhangs, which is a feature unique to GIY-YIG enzymes. Thus, in order to resolve this question, I have performed new biochemical and kinetic analyses of wild-type and mutant proteins that have had a conserved residue in one of the motifs substituted with alanine. The results indicate that not only does I-CreII have a functional GIY-YIG motif, but the data point to it as the catalytic domain. From this new perspective, this is also the first quantitative study of a GIY-YIG endonuclease. These data also provide the first direct evidence that an H-N-H motif can function primarily in DNA binding rather than catalysis- a finding that has implications for transcription factors from lower plants and protists that appear to have this motif.

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