Homing endonucleases are extraordinarily specific DNA-binding proteins, acting specifically at individualsites within a host genome. These proteins are under instense study for the purpose of engineering singlechain gene-specific reagents to be used for gene therapy and other applications. Over the past 10 years, wehave determined the structure and mechanisms of representatives form all known families of homingendonucleases, found respectively in phage, eubacteria, archae, and single cell eukarya. In addition, wehave described the creation of homing endonuclease variants that act at noncognate sites. These constructshave been generated using both bacterial selection strategies and compuational methods, both of whichtarget enzyme residues that directly contact DMA basepairs. In either case, such experiments have producedendonucleases that display shifted DMA recognition properties, but at the cost of reduced site-discriminationabilities. We hypothesize that in order to completely reprogram the DNA recognition specificity of a homingendonuclease, without a reduction in site discrimination, the resculpting of protein-DNA contacts must becombined 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 NorthwestGenome Engineering Consortium is to accomplish this task by combining somatic hypermutation of theendonuclease scaffold, computational redesign and selection of DNA contacts, and biochemical/biophysicalcharacterization 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 tworelated methods to directly visualize cleavage of DNA target variants and to quantitate specificity at eachbase pair.2. We will determine the thermodynamic signature of cognate and non-cognate site recognition forredesigned homing endonucleases, using isothermal titration calorimetry (ITC).3. We will determine the three-dimensional structure of novel endonuclease-DNA cognate pairs at highresolution, 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.
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