Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM

Abstract

Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts

“…Collectively, these results indicate that the scissile base pair (position 1) and its interactions with the other nucleotides are the most important determinant of recombination efficiency. This might, at least partly, be due to the central role that the scissile base plays in several steps of the recombination process, most importantly the formation of the synaptic complex and the positioning of the DNA strands 66 – 68 .…”

Synthetic genome modules designed for programmable silencing of functions and chromosomes

“…This chromatin compaction into a solenoid structure is known to facilitate transcriptional repression 66 . Given that Cre is known to self-assemble into tetramers 67 this suggests a potential for dCre to compact DNA upon its binding, possibly changing the 3D structure of chromosomal DNA in a manner analogous to the solenoid structure. Thus, chromosome conformation changes and histone modifications related to epigenetic silencing are avenues worth investigating further in the future.…”

Cre-loxP-mediated genetic lineage tracing: Unraveling cell fate and origin in the developing heart

“…Cre recombinase is a 38 kDa protein encoded using P1 bacteriophage, which is highly conserved and catalytically active and can efficiently achieve site-specific DNA fragmentation and recombination both in vivo and in vitro (24)(25)(26). Similarly, the locus of crossing over in phage P1 (27,28) is also derived from the P1 phage, composed of two 13 bp inverted repeats and an 8 bp sequence in the middle.…”