Title

Distance Determination by GIY-YIG Intron Endonucleases: Discrimination between Repression and Cleavage Functions

Document Type

Article

Publication Date

3-2006

Abstract

GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNAbinding 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.

Notes

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