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Nucleic Acids Research 2006 34(6):1755-1764; doi:10.1093/nar/gkl079
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Published online 31 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions

Qingqing Liu1,2, Victoria Derbyshire1, Marlene Belfort1,* and David R. Edgell1,3

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.


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Home page
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