Purification and In Vitro Characterization of Zinc Finger Recombinases.

Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance. Here, we present a straightforward set of protocols for the expression and purification of ZFRs, an assay system for catalytic proficiency in vitro and bandshift assays for detection of sequence-specific DNA interactions.

Intermediates in serine recombinase-mediated site-specific recombination.

Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.

Site-specific DNA recombinases as instruments for genomic surgery.

Site-specific DNA recombinases can "cut and paste" DNA. For example, they can promote excision of specific DNA segments or insertion of new DNA segments in specific places. However, natural recombinases act only at their cognate recombination sites, so current applications are limited to genetically modified organisms in which these sites have been introduced into the genome. Transposases also catalyze DNA rearrangements; they promote insertion of specific DNA sequences but at nonspecific locations. Applicability of site-specific recombinases and transposases in experimental genetics, biotechnology, and gene therapy would be much wider if they could be re-engineered so as to act specifically at chosen sequences within an organism's natural genome. This review will discuss progress towards the creation of such "designer" recombinases.