Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease.
We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or 'homing' site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA-protein interface. Biochemical characterization of these proteins revealed a wide range of site-binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.
Eklund
Jennifer L
JL
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Ulge
Umut Y
UY
Eastberg
Jennifer
J
Monnat
Raymond J
RJ
D004275:DNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D004275:DNA, Ribosomal; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease