DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI.
Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). 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 intron. 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 endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A 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.
Flick
K E
KE
Fred Hutchinson Cancer Research Center, University of Washington, Seattle 98109, USA.
Jurica
M S
MS
Monnat
R J
RJ
Stoddard
B L
BL
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism
D004268:DNA-Binding Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; D015032:Zinc