Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination

Abstract

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131–151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

Kinetic properties of optogenetic DNA editing by LiCre-loxP

“…Since, for LiCre, the steps of synapse assembly and catalysis are not expected to be faster (LiCre was derived from Cre by reducing, rather than increasing, its propensity to assemble the complex and achieve the reaction), the main difference between in vitro (Shoura et al, 2012) and in vivo (this study) R 0 values likely lies into the probability of forming a DNA loop bringing the two loxP sites together (a pre-requisite to synapse formation). This probability depends on the mechanical properties of the inter- loxP DNA segment (Rosa et al, 2010; Shoura et al, 2020); it may thus differ strongly between a stiff inter- loxP segment of naked dsDNA in vitro and a softer, chromatinized one in vivo (Hajjoul et al, 2013; Ringrose et al, 1999).…”

Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases

“…The difficulty to obtain experimentally structural information through X-ray crystallography about this Nt tail has so far precluded any detailed study on its possible involvement in DNA target recognition in Cre-like SSRs. Besides the extensive studies performed on this recombination system by X-ray crystallography [12] , several other biophysical experimental and computer-aided studies focusing on DNA topology, kinetics of DNA-loop formation and synapsis geometry have been directed towards acquiring information about the conformational dynamics, stability and topology of reaction intermediates and, thus, the recombination mechanisms of the Cre recombinase and related systems [33] , [34] , [35] , [36] , [37] . These studies have provided relevant structural, dynamic and functional mechanistic information on these recombinase systems and a complementary view to the one provided by X-ray crystallography.…”