Published online 31 March 2006
Article |
Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions
1 New York State Department of Health, Wadsworth Center, Center for Medical Science 150 New Scotland Avenue, Albany, NY 12208, USA 2 Department of Biological Sciences, State University of New York at Albany Albany, NY 12222, USA 3 Department of Biochemistry, University of Western Ontario London, ON N6A 5C1, Canada
*To whom correspondence should be addressed. Tel: +1 518 473 3345; Fax: +1 518 474 3181; Email: belfort{at}wadsworth.org
Received December 14, 2005. Revised February 1, 2006. Accepted March 4, 2006.
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.