Role of the interdomain linker in distance determination for remote cleavage by homing endonuclease I-TevI.
I-TevI is a modular intron-encoded endonuclease, consisting of an N-terminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus: the deletion-intolerant (DI) region; the deletion-tolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a beta-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the beta-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.
Liu
Qingqing
Q
Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.
Dansereau
John T
JT
Puttamadappa
Shadakshara S
SS
Shekhtman
Alexander
A
Derbyshire
Victoria
V
Belfort
Marlene
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease