Engineering Homing Endonucleases as Gene-Specific Reagents for Cancer Therapy Human cancer often results from the mutation and/or altered expression of specific genes. These altered genes provide specific molecular targets for the development of novel therapeutic agents that can selectively modify gene structure or expression in human cells. We will use a combination of structural, genetic and biochemical approaches to determine how two lower eukaryotic homing endonucleases, I-PpoI, and I-CreI, that bind and cleave long (15 and 24 base pairs, respectively) DNA target sites, can be altered to bind and/or cleave new DNA target sequences with high specificity in vitro and in vivo. These variant endonucleases would represent a new class of gene-specific reagents (GSRs) for use in human cells.
The basis for this is previous work in the sponsors' labs. The DNA-protein cocrystal structures of both endonucleases have been determined, endonuclease and homing site mutations that alter DNA cleavage have been isolated or constructed and characterized. We have further demonstrated that site-specific DNA binding and cleavage can be separated by specific mutations in the endonuclease active sites. I am curently using these results to develop and test atomic-level resolution models of the structural basis for DNA-protein recognition by both endonucleases. I will then use these models to design and construct variant endonucleases with the ability to bind long novel DNA target sites with high specificity. Engineering of these novel GSRs will employ a combination of site-directed mutagenesis and the partial randomization of endonuclease protein residues followed by genetic selection or screening. Endonuclease variants with the ability to bind long novel DNA target sequences will be further characterized using a combination of biochemical and structural approaches, and will be assayed for their ability to modify gene structure and expression in human cells.
This interdisciplinary project demands broad scientific and technical knowledge. The requisite expertise is provided by the collaboration between the Stoddard (structural biophysics) and the Monnat (pathology) laboratories.
Galburt, E.A., B. Chevalier, W. Tang, M.S. Jurica, K.E. Flick, R.J. Monnat and B.L. Stoddard. 1999. A novel endonuclease mechanism directly visualized for I-PpoI. Nature Structural Biology 6:1096-1099.