| Popis: |
Genome-editing (GE) is a form of genetic engineering that permits the deliberate manipulation of genetic material for the study of biological processes, agricultural and industrial biotechnologies, and developing targeted therapies to cure human disease. While the potential application of GE is wide-ranging, the efficacy of most strategies is dependent upon the ability to accurately introduce a double-stranded break at the genomic location where alterations are desired. LAGLIDADG homing endonucleases (LHEs) are a class of mobile genetic element that recognize and cleave 22-bp sequences of DNA. Given this high degree of specificity, LHEs are powerful GE reagents, but re-engineering their recognition sites has been hindered by a limited understanding of structural constraints within the family, and how cleavage specificity is regulated in the central target site region. In the present studies, a covariation analysis of the LHE family recognized a set of coevolving residues within the enzyme active site. These positions were found to modulate catalytic efficiency, and are thought to create a barrier to active site evolution and re-engineering by constraining the LHE fitness landscape towards a set of functionally permissive combinations. Interestingly, mutation of these positions led to the identification of a catalytic residue variant that demonstrates cleavage activity against a greater number of central target site substrates than wild-type enzymes. To facilitate these investigations, high-throughput and unbiased methods were developed to functionally screen large mutagenic libraries and simultaneously profile cleavage specificity against 256 different substrates. Lastly, structural studies aimed at increasing our understanding of the LHE coevolving network led to the discovery of direct protein-DNA contacts in the central target site region. Significantly, these findings increase our understanding of functionally important structural constraints within the LHE family and have the potential to increase the sequence targeting capacity of LHE scaffolds. More broadly, the methodologies described in this thesis can assist large-scale structure-function studies and facilitate investigations of substrate specificity for most DNA-binding proteins. Finally, the thorough biochemical validation I provide for computational predictions of coevolution showcases a strategy to infer protein function-structure from genetic information and emphasizes the need to expand these studies to other protein families. |
