No abstract

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.

(Fig. 1a).The attI site, like other site-specific recombinase binding sites, contains a core of ell as two sive towardsThe attC sites in contrast display poor sequence con gions of sequence similarity at their boundaries. These conserved regions are separated by a stretch of imperfect internal dyad symmetry 7 .One might have expected that excision of a gene cassette would occur via the classic model of Holliday junction (HJ) formation and resolution using two duplex attC sites as observed with Cre-mediated loxP recombination 10 (Fig. 1b) Structure of VchIntIA RecombinaseVchIntIA folds into two distinct domains ( le stranded attC substrates, we demonstrated that the bottom strand of attC recombined with a resident attI at a rate of 1000-fold higher than the comparable top strand of attC 12 .Disruption of the postulated 13-16 secondary structure of attC affected recombination.Based on these results we proposed a recombination model (Fig. 1c). The structure of the across the synaptic interface. This interaction allows helix I 2 from the atta unit(s) to form several important DNA contacts in trans holding the sThese trans-interactions mediated by G20'' result in only a two-fold symm synapse which may inhibit HJ isomerization as discussed below. Subunits 2 (Fig. 2b, 3). The spacing and geometry of the bases in the central region duplex preclude intersubunit interaction between the N-termini on the sam 2a, 3). The more extensive intersubunit interface across the synapse burie accessible surface area. These interfacial contacts are due namely the Integron Site-Specific RecombinationRecently, exploiting conjugation as a medium to exclusively deliver sing 9Hal-Pasteur author manuscript pasteur-00140781, version 1VchIntIA-VCR bs complex presented here provides a structural basis for IntI mediated site-specific recombination using the bottom strand of attC as a substrate. (Fig. SF6a).This suggests a higher occupancy of only one half site and change in the effective utated ty (Fig. SF6c).I and W219I), also ision frequency. These results suggest that G20'' plays a central role in maintaining an active VchIntIA-VCR bs synapse, but that other factors namely C-terminal helix exchange and B/C and A/D interfaces also contribute to the assembly of the tetrameric synapse.A folded single-strand attC substrate during integron recombination necessitates that second strand cleavage and transfer is down-regulated relative to most members ofThe biological relevance of the observed structural aspects of our m cis-and trans-interactions observed in the VchIntIA-VCR bs complex, w using a combination of EMSA and in vivo excision assays 13 . Four di observed with wild type VchIntIa-VCR (SF6a), potentially correspond however, only a 5-fold drop in excision frequency (Fig. S 6c). Mutagene (Fig. 1c).In addition the rotation (~15 o ) of the C-terminal domains within the non-attacking subunits could also reduce the rate of second strand cleavage (Fig. SF8). This movement and flanking (R' and R'') repeats. The nucleotides, T12'' (red) and G20'...