Homing endonucleases are extraordinarily specific DNA-binding proteins, acting specifically at individual sites within a host genome. These proteins are under instense study for the purpose of engineering single chain gene-specific reagents to be used for gene therapy and other applications. Over the past 10 years, we have determined the structure and mechanisms of representatives form all known families of homing endonucleases, found respectively in phage, eubacteria, archae, and single cell eukarya. In addition, we have described the creation of homing endonuclease variants that act at noncognate sites. These constructs have been generated using both bacterial selection strategies and compuational methods, both of which target enzyme residues that directly contact DMA basepairs. In either case, such experiments have produced endonucleases that display shifted DMA recognition properties, but at the cost of reduced site-discrimination abilities. We hypothesize that in order to completely reprogram the DNA recognition specificity of a homing endonuclease, without a reduction in site discrimination, the resculpting of protein-DNA contacts must be combined with the selection of structural mutations in the nearby enzyme scaffold that """"""""fine-tune"""""""" the protein -DNA interaface of each novel cognate complex. The goal of overall Specific Aim 1 of the Northwest Genome Engineering Consortium is to accomplish this task by combining somatic hypermutation of the endonuclease scaffold, computational redesign and selection of DNA contacts, and biochemical/biophysical characterization of the resulting endonuclease constructs. In our component of the consortium's activities, we will be responsible for the following aims: 1. We will determine the in vitro site specificity profile of the novel endonuclease construcst using two related methods to directly visualize cleavage of DNA target variants and to quantitate specificity at each base pair. 2. We will determine the thermodynamic signature of cognate and non-cognate site recognition for redesigned homing endonucleases, using isothermal titration calorimetry (ITC). 3. We will determine the three-dimensional structure of novel endonuclease-DNA cognate pairs at high resolution, and will characterize (a) the effect of enzyme scaffold mutations on backbone structure, and 9b) the accuracy of computational redesign predictions within the protein-DNA interface.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Special Emphasis Panel (ZRR1-SRC (99))
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Knowlton, John R
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Fred Hutchinson Cancer Research Center
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Baxter, Sarah K; Scharenberg, Andrew M; Lambert, Abigail R (2014) Engineering and flow-cytometric analysis of chimeric LAGLIDADG homing endonucleases from homologous I-OnuI-family enzymes. Methods Mol Biol 1123:191-221
Chan, Yuk-Sang; Takeuchi, Ryo; Jarjour, Jordan et al. (2013) The design and in vivo evaluation of engineered I-OnuI-based enzymes for HEG gene drive. PLoS One 8:e74254
Metzger, Michael J; Stoddard, Barry L; Monnat Jr, Raymond J (2013) PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks. DNA Repair (Amst) 12:529-34
Mak, Amanda Nga-Sze; Bradley, Philip; Bogdanove, Adam J et al. (2013) TAL effectors: function, structure, engineering and applications. Curr Opin Struct Biol 23:93-9
Jacoby, Kyle; Metzger, Michael; Shen, Betty W et al. (2012) Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space. Nucleic Acids Res 40:4954-64
Baxter, Sarah; Lambert, Abigail R; Kuhar, Ryan et al. (2012) Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases. Nucleic Acids Res 40:7985-8000
Taylor, Gregory K; Petrucci, Lucas H; Lambert, Abigail R et al. (2012) LAHEDES: the LAGLIDADG homing endonuclease database and engineering server. Nucleic Acids Res 40:W110-6
Stoddard, Barry L (2011) Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 19:7-15
Chan, Siu-Hong; Stoddard, Barry L; Xu, Shuang-Yong (2011) Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity. Nucleic Acids Res 39:1-18
Takeuchi, Ryo; Lambert, Abigail R; Mak, Amanda Nga-Sze et al. (2011) Tapping natural reservoirs of homing endonucleases for targeted gene modification. Proc Natl Acad Sci U S A 108:13077-82

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