Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions.
GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNA-binding domains. I-TevI, the T4 phage GIY-YIG intron endonuclease, functions both in promoting td intron homing, and in acting as a transcriptional autorepressor. Repression is achieved by binding to an operator, which is cleaved at 100-fold reduced efficiency relative to the intronless homing site. The linker includes a zinc finger, which functions in distance determination, to constrain the catalytic domain to cleave the homing site at a fixed position. Here we show that I-BmoI, a related GIY-YIG endonuclease lacking a zinc finger, also possesses some cleavage distance discrimination. Furthermore, hybrid endonucleases constructed by swapping the domains of I-BmoI and I-TevI are active, precise and demonstrate that features other than the zinc finger facilitate distance determination. Most importantly, I-TevI zinc finger mutants cleave the operator more efficiently than the homing site, the converse of wild-type protein. These results are consistent with the zinc finger acting as a measuring device, directing efficient cleavage of the homing site to promote intron mobility, while reducing cleavage at the operator to ensure transcriptional autorepression and phage viability.
New York State Department of Health, Wadsworth Center, Center for Medical Science, 150 New Scotland Avenue, Albany, NY 12208, USA.
Qingqing
Q
Liu
Victoria
V
Derbyshire
Marlene
M
Belfort
David R
DR
Edgell
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease; C065074:I-TEVI endonuclease