Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Modular Assembly of a Multi-functional Homing Endonuclease

Homing endonucleases are usually encoded within self-splicing introns or inteins, and are found in all three biological kingdoms. The endonuclease genes and their host intron or intein are composite selfish genetic elements that spread between related genomes by a process called homing. The homing pathway is initiated by the endonuclease binding to a sequence, the homing site, centered on the intron insertion site (IS) of intronless alleles. The role of the homing endonuclease is limited to introduction of a double-strand break (DSB) close to the intron IS.

I-TevI, a member of the GIY-YIG family of homing endonucleases, is a site-specific yet sequence tolerant enzyme. It is a remarkably extended molecule consisting of a well-folded catalytic domain connected to a series of sub-domains by extended regions (1, 2). These include a Zn-finger, a minor-groove binding helix and a helix-turn-helix subdomain. The modular nature of the enzyme is pertinent to the evolution of homing endonucleases. We have begun to assess the role of each subdomain in endonuclease function. Most interestingly, we have found that the Zn-finger subdomain does not play a role in substrate DNA binding. Rather it is required for positioning the catalytic domain at a set distance from the primary binding site for cleavage to occur (3). The next challenge is trying to understand the nature of the linker that connects the catalytic and DNA-binding domains and how it interacts with the Zn-finger to mediate distance determination.

In addition, recent work has shown that the enzyme is bi-functional and can act as an auto-repressor (4). It can bind to a site present in its promoter region that exhibits sequence similarity to its homing site, but does not cleave the DNA. It is the very modular nature of the enzyme, with distinct sequence requirements for binding and cleavage, that facilitates its bifunctionality by binding in the absence of cleavage, leading to transcriptional repression.

References and Footnotes
  1. Van Roey, P., Waddling, C. A., Fox, K. M., Belfort, M., and Derbyshire, V. EMBO J. 20, 3631-3637 (2001).
  2. Van Roey, P., Meehan, L., Kowalski, J. C., Belfort, M. and Derbyshire, V. Nat. Struct. Biol. 9, 806-811 (2002).
  3. Dean, A. B., Stanger, M. J., Dansereau, J. T., Van Roey, P., Derbyshire, V., and Belfort, M. Proc. Natl. Acad. Sci. USA 99, 8554-8561 (2002).
  4. Edgell, D. R., Derbyshire, V., Van Roey, P., LaBonne, S., Stanger, M. J., Li, Z., Boyd, T. M., Shub, D. A., and Belfort, M. Nat. Struct. Mol. Biol. 11, 936-944 (2004).

Marlene Belfort1
Patrick Van Roey
Victoria Derbyshire1,*

Wadsworth Center
New York State Department of Health
1State University of New York at Albany
P.O. Box 22002
Albany, NY 12201-2002

*Email: vicky.derbyshire@wadsworth.org