Structural and mechanistic studies of the Type IIS restriction endonuclease PaqCI and de novo designed circular tandem repeat proteins

Abstract

This thesis spans the work completed on two parallel projects, to (i) study the structure and mechanism of the Type IIS restriction endonuclease PaqCI, and (ii) participate in the characterization and engineering of de novo designed circular tandem repeat proteins (‘cTRPs’) for the development of novel ligand-dependent protein dimerization systems. Through the latter project, I gained early intensive training in protein crystallography and engineering, contributing to my independent study of PaqCI. Restriction endonucleases are an essential component of innate, 'preprogrammed' phage restriction systems that protect bacteria from foreign DNA. Type II restriction endonucleases are invaluable tools in research because of their ability to identify and cleave specific DNA sequences with extremely high fidelity, as well as their unique mechanisms of cleavage. The most well-studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or related enzymes form a DNA-bound reaction complex have not been described at atomic resolution. Here I describe a series of crystallographic and CryoEM structures in the presence and absence of bound DNA targets that reveal aspects of DNA recognition and cleavage by the Type IIS PaqCI restriction endonuclease. The structures illustrate the enzyme's tetrameric domain organization in the absence of bound substrate and the subsequent formation of a tetrameric reaction complex poised to deliver the first of a series of double-strand breaks. Understanding the structure of the Type IIS restriction endonucleases PaqCI reveals (i) the requirement for multiple DNA targets to be pulled together for enzyme activation, (ii) that enzymatic domains are sterically constricted and can only correctly orient for cleavage at 4/8 bases from the target site, and (iii) that the orientation of the target recognition domain on the DNA determines the motion required of the endonuclease domains to engage the cleavage site. These results bolster the dominant hypothesis that Type II restriction enzymes require the engagement of multiple unmodified targets to bias cleavage towards unprotected foreign DNA. Through these two projects I gained expertise in crystallography and cryoEM, enzymatic analyses, and protein engineering.