Abstract
The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-specific recombinases (SSRs) for genome editing applications. Previous work has revealed key residues for target site selectivity in the Cre/loxP and the related Dre/rox recombinase systems. However, enzymes in which these residues were changed to the respective counterpart only showed weak activity on the foreign target site. Here, we use molecular modeling and dynamics simulation techniques to comprehensively explore the mechanisms by which these residues determine target recognition in the context of their flanking regions in the protein–DNA interface, and we establish a structure-based rationale for the design of improved recombination activities. Our theoretical models reveal that nearest-neighbors to the specificity-determining residues are important players for enhancing SSR activity on the foreign target site. Based on the established rationale, we design new Cre variants with improved rox recombination activities, which we validate experimentally. Our work provides new insights into the target recognition mechanisms of Cre-like recombinases and represents an important step towards the rational design of SSRs for applied genome engineering.
Highlights
Site-specific DNA recombinases (SSRs) are powerful tools for precise DNA rearrangements to allow inversions, deletions and translocations in the genome of heterologous h osts[1,2,3,4]
Utilizing structure-based modeling and molecular dynamics (MD) simulations, we explored the interactions involved in protein-DNA binding in these complexes, at the interface formed by the specificity-determining amino acids at positions 43, 259 and 263, which lay on helix B and J, and the nucleotides at positions 10/66, 11/65, and 12/64 (Fig. 1b,c)
To better understand how Cre-type site-specific DNA recombinases may achieve better precision in terms of recombination activity, we investigated in detail the protein-DNA recognition properties of such recombinase systems in a comparative fashion by applying molecular modeling and dynamics simulations
Summary
Site-specific DNA recombinases (SSRs) are powerful tools for precise DNA rearrangements to allow inversions, deletions and translocations in the genome of heterologous h osts[1,2,3,4]. The discovery of several such systems, including the Dre/rox[9], VCre/VloxP10, SCre/SloxP10, Vika/vox[11], Nigri/nox[12] and Panto/pox[12] recombinase systems has greatly expanded the repertoire of available SSRs that can be used alone or in combination to allow advanced genome e ngineering[13,14,15], and to build sophisticated synthetic biology c ircuits[16,17]. While these enzymes, as well as their target sites, share high sequence similarities, cross-recombination is typically not observed. In order to establish a structure–function rationale, which could help in guiding further efforts for improving recombination properties, we decided to investigate in detail the molecular recognition mechanisms of these specificity-determinant key residues at positions 43, 259 and 263 in binding to loxP and rox sites
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