Abstract
Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns1,2,3. They have also been identified in self-splicing inteins (protein introns)4. These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron3. The target sites recognized by these small endonucleases are generally long (14–44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His–Cys box, the GIY–YIG and the H–N–H endonucleases1. The first identified His–Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum5,6. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility7. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 Å resolution. I-PpoI displays an elongated fold of dimensions 25 × 35 × 80 Å, with mixed α/β topology. Each I-PpoI monomer contains three antiparallel β-sheets flanked by two long α-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 Å apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.
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Acknowledgements
We thank D. McHugh, K. Stephens and J. D. Heath for initial subcloning, purification and crystallization studies; R. Strong, K. Zhang and B. Scott for advice during the crystallographic analysis; and the beamline staff at the Advanced Light Source (NLBL laboratories), beamline 5.0.2, particularly T. Earnest, for assistance. B.L.S. and R.J.M. are funded for this project by the NIH. K.E.F. was supported by an NIH training grant and the American Heart Associaiton. M.S.J. was supported by an NSF fellowship and an NIH training grant.
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Flick, K., Jurica, M., Monnat, R. et al. DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature 394, 96–101 (1998). https://doi.org/10.1038/27952
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DOI: https://doi.org/10.1038/27952
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