A chimeric Cre recombinase with regulated directionality

Abstract

From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long-and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.bacteriophage ͉ recombinase ͉ site-specific recombination ͉ evolution

Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination

“…Our strategy for genetically testing the Int bridges in a full recombination reaction depends on a previously constructed and characterized chimeric recombinase in which the amino-terminal domain of λ-integrase was fused with the Cre recombinase (17). Here we refer to this recombinase as Crn1.…”

Cre Recombinase

“…The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts (15), has been an important factor in both cases. Cre has also been used in experiments designed to understand the functions of other recombination systems (16)(17)(18).…”

Teaching Cre to follow directions

“…An article in a recent issue of PNAS (4) provides important new insights into both questions. By making a chimeric protein composed of Cre recombinase fused to a small DNAbinding domain of -integrase, Warren et al (4) have turned the normally unregulated Cre into a regulated integrase with the same requirements and directionality found in the -integrase system.…”

“…Because the presence of an armbinding N-domain is the most obvious difference between the Cre and -integrase proteins, Warren et al (4) asked whether adding an N-domain to Cre would lead to a novel recombinase with the regulatory properties of -integrase. To test this idea, they generated modified attP and attB sites for integration (called lotP and lotB) and modified attL and attR sites for excision (called lotL and lotR) in which the core -integrase binding sites were replaced by weakened Cre binding sites.…”

“…The first could occur by simple mutation of the recombinase and/or the binding sites. The second could occur as the outcome of homologous recombination events, but Warren et al (4) point out that an alternative, more efficient mechanism could also play an important role. Many recombinase genes are located adjacent to their att sites, setting the stage for fusion with new sequences via site-specific recombination with weak att sites located within the recombinase gene and elsewhere in the bacterial genome.…”