Structural and biochemical studies of site-specific recombinases.

摘要

Site-specific recombinases catalyze DNA rearrangements in a variety of biological processes and they have been utilized in many in vitro and in vivo genome engineering applications. Most recombinases can be grouped into two distinct families, the tyrosine and serine recombinases. My thesis work has been primarily investigating the structure and mechanism of the serine integrases, which have been recently demonstrated to function in a variety of organisms including human cells. As part of an overall effort to develop structural views on recombination by the serine integrases, I have determined the crystal structure and solution properties of the N-terminal catalytic domain from bacteriophage TP901-1 integrase. The crystal structure reveals a symmetric tetramer representing a unique intermediate along the recombination pathway. The overall organization serves as the first structural evidence that the serine integrases utilize a DNA-outside architecture. Analytical ultracentrifugation and small-angle X-ray scattering (SAXS) results have provided further views of the synaptic tetramer formation from a pair of solution dimers. The C-terminal domain (CTD) of the serine integrases is expected to contribute tremendously to their unique recombination features. I have produced diffraction-quality co-crystals of Listeria innocua integrase CTD bound to an attP half-site. Structure determination of the CTD-DNA complex would reveal critical information on specific sequence recognition and site-selectivity mechanisms. Furthermore, I have obtained initial crystals of a full-length integrase bound to a full attP site that have shown promising diffraction. Together, the results are a significant start towards developing a structural framework for understanding the recombination mechanism by the serine integrases.;The remainder of my thesis work has focused on the structural and functional studies of Cre recombinase from the tyrosine recombinase family bound to an isolated loxP half-site. Here I report the crystal structure of Cre complexed with a half-site loxP DNA substrate. This is the first structural view of a monomeric Cre-DNA complex and represents a unique structural snapshot that contributes to understanding the complex Cre-lox assembly process. Site-directed crosslinking experiments and concentration dependent half-site cleavage assays have provided additional mechanistic views on Cre-loxP interaction.