Cre Recombinase and Other Tyrosine Recombinases

Abstract

Tyrosine-type site-specific recombinases (T-SSRs) have opened new avenues for the predictable modification of genomes as they enable precise genome editing in heterologous hosts. These enzymes are ubiquitous in eubacteria, prevalent in archaea and temperate phages, present in certain yeast strains, but barely found in higher eukaryotes. As tools they find increasing use for the generation and systematic modification of genomes in a plethora of organisms. If applied in host organisms, they enable precise DNA cleavage and ligation without the gain or loss of nucleotides. Criteria directing the choice of the most appropriate T-SSR system for genetic engineering include that, whenever possible, the recombinase should act independent of cofactors and that the target sequences should be long enough to be unique in a given genome. This review is focused on recent advancements in our mechanistic understanding of simple T-SSRs and their application in developmental and synthetic biology, as well as in biomedical research.

Xer Site Specific Recombination: Double and Single Recombinase Systems

“…HJ formation and isomerization activates the second pair of subunits bound to the other half of the recombination sites and inactivates the first pair of subunits. The second pair of subunits then cleaves, exchanges and rejoins the second pair of strands by the same mechanism just described; this second cleavage allows the resolution of HJ-intermediate and results in the recombinant DNA ( Figure 2B ) (Rajeev et al, 2009; Meinke et al, 2016). This process implies that the specific pairs of recombinases and/or active sites are continuously switched on and off to synchronize when and how recombination occurs, this coordination depends on allosteric interactions between the recombinases and external factors imposed on the synaptic complex (Hallet et al, 1999; Aussel et al, 2002; Vanhooff et al, 2009).…”

“…The synaptic XerCD/ dif complex consists of two XerC and two XerD subunits respectively bound to two dif sites ( Figure 2B ). Limited structural information of some tyrosine recombinases have revealed a conserved catalytic domain fold (Swalla et al, 2003), facilitating the analysis of experimental data and allowing the development of a general model for Xer recombinases (Meinke et al, 2016) consisting of; XerD (Subramanya et al, 1997), XerA (Hwa Jo et al, 2016), XerH (Bebel et al, 2016) and other related tyrosine recombinases like Cre (Gopaul and Van Duyne, 1999; Martin et al, 2002), HP1 integrase (Hickman et al, 1997), FLP (Chen et al, 2000) and λ integrase (Kwon et al, 1997; Biswas et al, 2005). The E. coli dif site is divided into two 11 bp half-sites that share partial dyad symmetry linked by a 6 bp central region that defines the positions of strand cleavage and exchange (Kuempel et al, 1991).…”

“…Subsequent investigations led to the identification of SSR enzymes involved in dimer resolution in other plasmids of E. coli , and other bacteria. Current estimates have identified more than 1300 tyrosine recombinases where many of them are associated with other host proteins to regulate their activity, directionality, or processivity (Meinke et al, 2016). Large plasmids usually carry their own recombinase machineries adjacent to the recombination site.…”

“…Indeed, initial studies on plasmid stability in ColE1 and phage integration of bacteriophage λ led to the discovery of XerC and the mechanistic insights of the tyrosine family (Meinke et al, 2016). In V. cholerae , the causative agent of the potentially fatal human disease cholera, XerC V C and XerD V C are hijacked by some vibriophages to integrate their genomes into the chromosome.…”

Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

“…This approach uses Cre recombinase from bacteriophage P1 to carry out site-specific recombination between two DNA recognition sites, known as LoxP sites, allowing precise manipulation of genomes, and has been used widely in mouse genetics [31]. Cre target specificity can be manipulated to generate novel site-specific recombinases via directed evolution [32] using substrate-linked protein evolution (SLiPE), which places the recombination target site of interest adjacent to the recombinase coding region, allowing DNA molecules with a successful recombinase coding region to be marked on the linked substrate DNA and recovered from the background of unsuccessful recombinases by PCR [32].…”