1. Reciprocal Control of Catalysis by the Tyrosine Recombinases XerC and XerD 
Bernard Hallet, Lidia K Arciszewska, David J Sherratt 
Molecular Cell 
Volume 4, Issue 6, Pages (December 1999)
DOI: /S (00)

2. Figure 1 Recombinase Sequences and Structures
(A) Alignment of the primary and secondary structures of the C terminus of XerC, XerD, and Cre. Conserved motif II is boxed. Catalytic residues are circled with yellow. The divergent ESS–NHG tripeptide of XerC and XerD and the equivalent RAG tripeptide of Cre are highlighted in blue. Position of XerD and Cre α helices is shown by cylinders. Dotted lines underline disorded residues.
(B) Structure of XerD (Subramanya et al. 1997) and of the covalent Cre–DNA synaptic complex in which two of the four subunits (in purple) have cleaved the DNA to form a 3′ phosphotyrosine linkage (Guo et al. 1997). The phosphate groups of the cleaved DNA strands are shown in yellow. The N-terminal domains of both recombinases, which in the Cre complex form a tetrameric platform on the opposite face of the synapse, have been omitted for clarity. The C-terminal α helices L, M, and N of XerD and α helices K, L, M, and N of Cre are represented by cylinders. The NHG and RAG tripeptides are highlighted in a blue surface representation.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2
Altered Reciprocal Patterns of Strand Exchange on Different HJ Substrates
(A) The direction of HJ resolution by XerC and XerD is determined by the propensity of the substrate to adopt a conformation in which either the top strands (thick lines) or the bottom strands (thin lines) are crossing. All possible combinations of the wild-type XerC (WT), XerCY275F (YF), XerCNHG (NHG), or XerCNHG/Y275F (NHG-YF), and the wild-type XerD (WT), XerDY279F/Y279F (YF), XerDESS (ESS), or XerDESS/Y279F (ESS-YF) were incubated with the untethered dif, psi, and dif(GATCCA) HJ substrates. Sequences of the recombination sites central region are shown in brackets, with the purines in bold. After 30 min incubation at 37°C, the reactions were analyzed on 10% polyacrylamide gels containing 0.1% SDS. Resolution of the substrates by XerC or XerD generates radiolabeled (*) strand exchange products of different lengths (C and D, respec
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3
Altered Patterns of DNA Strand Cleavage on Linear Recombination Site Substrates
Radiolabeled (*) dif and psi suicide substrates containing a nick in the top strand were incubated for 60 min at 37°C with the indicated mixtures of recombinases. Reactions were analyzed by electrophoresis through 6% polyacrylamide gels containing 0.1% SDS. Position of the substrate band and of the radiolabeled XerC and XerD cleavage products is indicated. No radiolabeled XerC–half site complexes arising from double cleavage by XerC and XerD are detected. In the dif reactions, smearing bands in the expected region of the gel are proteolytic degradation products of the singly cleaved XerC–DNA complex.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4 Effects of IPSS Mutants on psi Recombination In Vitro
(A) Recombination between two psi sites (triangles) on pSDC134 plasmid produces two circles of 1.65 and 3.05 kb, respectively. Recombination takes place with a defined order in which XerC acts first to generate a HJ intermediate that is then resolved by XerD. Topological features of the DNA substrate and products are not shown for clarity.
(B) Supercoiled pSDC134 DNA was incubated for 60 min at 37°C with PepA and the indicated combinations of recombinases. DNA was digested with HindIII prior to electrophoresis on a 1% agarose gel. Bands are identified as follows: S1 and S2, small (1 kb) and large (3.7 kb) substrate fragments; HJ, HJ-containing χ structures; P1 and P2, linearized small and large resolution products.
(C) Similar analysis of 16 hr reactions performed with XerD variants in a buffer containing 10% glycerol (odd lanes) or 40% glycerol (even lanes).
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
Opposed and Synergetic Effects of XerDESS and C-Terminally Deleted Derivatives of XerC
(A) Resolution of dif and variant dif HJ substrates by XerCΔ10 and XerD. Display of the results is as in Figure 2A, except that the relative size of the XerC and XerD resolution products is reversed because a long arm of the HJ was 5′ labeled instead of a short arm.
(B) Resolution of the dif and dif(GATCCA) HJ substrates was examined by combining the wild-type XerD or XerDESS with XerC, XerCΔ5, or XerCΔ10. Labeling of the substrates was as in Figure 2A.
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6 Control of Catalysis in Xer Recombination
(A) Proposed reconfiguration of XerD C terminus upon assembly of the recombination complex on DNA. Left, schematic side view of XerD C-terminal domain based on the crystal structure of XerD (Subramanya et al. 1997). Right, the proposed donor–acceptor interactions between XerC and XerD and the folding of the recombinases C terminus are modeled according to the Cre–DNA crystal structures (Guo et al. 1997, Guo et al. 1999; Gopaul et al. 1998). Position of XerD catalytic residues (in red) and NHG tripeptide (in blue) is indicated. XerD target DNA phosphate is shown by a yellow circle. Only the extreme C terminus of the XerC subunit in the front right-hand position of heterotetramer is shown.
(B) A model for the sequential activation and reciprocal inhibition of pairs of recombinase active sites during Xer recombination. Representation of the XerC–XerD–DNA complex is based on the structure of the different Cre recombination intermediates. Color code is as in (A). The ball-and-socket joint depicts the interaction between the donor and acceptor regions of adjacent subunits. Step i to step v is the recombination pathway in which XerC strand exchange occurs first. (i) Interactions between XerC and XerD molecules bound on a same duplex, possibly coupled with additional interpromoter interactions across the synapse, force the DNA to bend in a configuration where the top (green) strand of the recombination site central region is exposed toward the outside of the duplex. The torsion energy stored in the bent DNA may act on the XerC–XerD donor–acceptor interaction so as to activate XerC catalysis by repositioning of the tyrosine nucleophile (arrowhead), and possibly other catalytic residues with respect to the DNA target phosphate (circle). DNA torsion strains released upon cleavage may also promote the unwinding and extrusion of the cleaved strands in order to orient the 5′ OH ends for the rejoining step. (ii) Completion of the strand exchange reaction generates a 2-fold symmetric HJ intermediate in which the top strands are crossing. (iii) Coupled protein and DNA conformation changes convert the complex into a configuration in which the bottom strands (purple) are crossing. (iv) This leads to synchronized inactivation of the XerC subunits and concomitant activation of the XerD subunits. (v) The recombinant duplexes are bent in the opposite direction to that of the initial recombination sites. This inversion of the DNA bending strains may promote the restacking of the DNA helices and the dissociation of the resealed molecules from the complex.
Molecular Cell 1999 4, DOI: ( /S (00) )