| The focus of my dissertation research has been to understand the basic biophysical properties of LAGLIDADG homing endonucleases at the atomic level, and to use this knowledge to engineer new enzymes that bind and cleave novel DNA target sites. First, I elucidated the LAGLIDADG catalytic mechanism by trapping I-CreI bound to its DNA both before and after cleavage. This reveals the two active sites to contain three metals, the central one of which is shared between both sites. Furthermore, the protein makes no direct contact to any member of the reaction pathway, including the nucleophilic waters, the scissile phosphates, nor the leaving groups. These results not only provide an understanding of the nuclease mechanism, but they suggest a rationale for the diversity of LAGLIDADG active site architecture. To further characterize the LAGLIDADG:DNA interface, I determined the atomic structures of two divergent isoschizomeric endonucleases bound to their DNA substrates. Comparison of the cocrystal structures reveals the binding specificity of these enzymes to be governed by strikingly different and unique sets of protein/DNA contacts; surprisingly, even most of the relatively few residues suggested by sequence alignments to be conserved are not. Indeed, of the approximately fifty residues that contact the DNA at the interface, only ten are conserved in both structures, and only four of these make base-specific contacts. Together, these interfaces offer insights into how LAGLIDADG proteins evolve to target new genomic sites. Finally, I used the knowledge gained in the above experiments to design a novel endonuclease. This enzyme, which we call E-DreI, contains elements from distantly related proteins (with a computationally redesigned domain interface). It is specific for its own target site and does not cleave the target sites of its ‘parent’ enzymes. The structure of E-DreI bound to DNA validates our experimental strategy by demonstrating the accuracy of the protein interface redesign algorithm and revealing how catalytic function is maintained during the creation of the new endonuclease. |
