Spatially-Directed Assembly of a Heterotetrameric Cre-Lox Synapse Restricts Recombination Specificity

SUMMARY

The pseudo-fourfold homo-tetrameric synapse formed by Cre protein and target DNA restricts site-specific recombination to sequences containing dyad-symmetric Cre-binding repeats. Mixtures of engineered altered-specificity Cre monomers can form heterotetramers that recombine non-identical asymmetric sequences, allowing greater flexibility for target site selection in the genome of interest. However, the variety of tetramers allowed by random subunit association increases the chances of unintended reactivity at non-target sites. This problem can be circumvented by specifying a unique spatial arrangement of heterotetramer subunits. By reconfiguring inter-subunit protein-protein contacts, we directed the assembly of two different Cre monomers, each having a distinct DNA sequence specificity, in an alternating (ABAB) configuration. This designed heterotetramer preferentially recombined a particular pair of asymmetric Lox sites over other pairs, whereas a mixture of freely-associating subunits showed little bias. Alone, the engineered monomers had reduced reactivity towards both dyad-symmetric and asymmetric sites. Specificity arose because the organization of Cre-binding repeats of the preferred substrate matched the programmed arrangement of the subunits in the heterotetrameric synapse. Applying this “spatial matching” principle, Cre-mediated recombination can be directed to asymmetric DNA sequences with greater fidelity.

INTRODUCTION

Controlled protein oligomerization underlies specificity in many biological processes. Multi-subunit complex assembly provides for precise targeting of binding or enzymatic activities as well as regulation of those activities through allosteric interactions and proximity effects. For sequence-specific binding proteins and enzymes, oligomerization of nucleic-acid interacting domains increases recognition site size and target specificity. The larger sites reduce the chance occurrences of recognition sequences elsewhere in the host genome, thereby minimizing detrimental off-target effects. For homo-oligomeric proteins, this increased specificity requires no extra protein coding capacity but limits recognition to repeated elements. Hetero-oligomeric assemblies can expand the range of possible target sequences by combinatorial mixing of differently-specific partners, as is the case with homeodomain, bZIP coiled-coil and bHLH transcription factors^1,2. However, the increased number of potential recognition sequences necessarily reduces the uniqueness of binding interactions. Specificity is achieved by a variety of mechanisms including regulated protein expression and localization, or stabilization of individual subunit combinations^3–6. Here, we enforced pairing of two variant tyrosine site-specific recombinase (YSSR) subunits in a defined spatial arrangement in order to direct recombination to specific asymmetric substrates, thus eliminating the requirement for repeated recognition sites while retaining a narrow specificity.

YSSRs efficiently induce crossovers between 20–40 bp DNA target sequences. In nature, the resulting integrations, excisions, inversions, and translocations are employed in chromosome and plasmid segregation, plasmid amplification, virus integration, and gene regulation^7–10. In biotechnology, YSSRs have been harnessed to manipulate chromosome structure in living cells and organisms to generate controlled gene deletions, integrate transgenes, and excise viral DNA^11–16. However, the high sequence specificity of the naturally-occurring YSSRs and the relaxed specificity of engineered versions remain an obstacle for the wide-spread use in applications requiring both flexible recognition potential and extreme precision, such as gene therapy¹⁷. One critical limitation is the homotetrameric nature of recombination complexes, which restricts recombination to occurring between nearly-identical, dyad-symmetric DNA sequences.

One well-studied YSSR, bacteriophage P1 Cre protein, carries out site-specific recombination at 34 bp LoxP sites^18–21. Cre-LoxP recombination is widely utilized for genome manipulations because of its robust activity and simple requirements. Numerous biochemical, biophysical and structural investigations have generated paradigms for YSSR recombination mechanism and function^22,23.

Recombination begins by assembly of a synaptic complex containing four Cre monomer subunits and two LoxP sites (Figure 1a)²⁴. As is typical for YSSRs, LoxP sites contain an inverted dyad of Cre-binding 13 bp repeats, separated by an asymmetric intervening sequence, the 8 bp spacer (Figure 1b)¹⁹. In the complex, “crossing-over” recombination results from two pairs of single-strand exchanges, effected by reversible cleavages and strand swaps within the 8 bp spacers (Figures 1a and 1b). The first exchange forms a Holliday junction (HJ) intermediate and the second exchange forms the products²¹. The order and progression of strand exchanges are directed by interplay between 8 bp spacer asymmetry, an associated DNA bend, and differentiation of Cre monomers into cleaving and non-cleaving conformations. This aspect of Cre function has been extensively studied and reviewed elsewhere^25–28.

Figure 1. Cre-LoxP recombination.

(a) YSSR recombination structural mechanism.²². Cre monomers are depicted as circles, and the two LoxP DNA strands are explicitly shown. LoxP directionality, as conventionalized in (b), is indicated by the pointed end. The critical CTH-CTD contact is depicted by the “ball-and-socket” connection between subunits (see (c)). The CTH is indicated by the small filled circle, the linker peptide is indicated by the connecting line, and the CTH docking site is indicated by the circular void. After complex assembly (left complex), two consecutive single-strand exchanges are carried out through cleavage/ligation events within the 8 bp spacer. The intervening 3′-phosphotyrosine covalent protein-DNA intermediates are not shown. The first exchange creates a Holliday junction (HJ) intermediate (center complex). The second strand exchange resolves the HJ to form the products, essentially as a reversal of the first exchange.

(b) LoxP. 34 bp LoxP sites consist of a dyad of “left arm” and “right arm” Cre-binding 13 bp repeats (blue boxes) flanking an asymmetric 8 bp “spacer” that contains the sites of DNA cleavage and ligation (black arrowheads). The convention for site directionality is indicated by the grey arrow as pointing from left arm to right arm, and all subsequent figures follow this convention, as indicated by the arrowheads. The LoxM7 variant contains three base substitutions in each of the 13 bp repeats (red boxes). In all subsequent figures, blue indicates a LoxP 13 bp repeat and red indicates a LoxM7 13 bp repeat. The CreALSHG variant, obtained through directed evolution (“C2(+/−) #4” in reference ³³), prefers to recombine LoxM7 compared to LoxP, whereas CreWT does not recombine LoxM7. Chimeric sites contain one LoxP and LoxM7 13 bp repeat each. LoxPM7 contains a LoxP left arm repeat and a LoxM7 right arm repeat, while LoxM7P contains a LoxM7 right arm repeat and a LoxP left arm repeat. CreWT also does not recombine these sites³⁴.

(c) The Cre-Lox recombination complex. The complex (PDB# 1CRX)²⁴, viewed from the C-terminal domain face, contains four Cre molecules and two Lox sites. With respect to the 8 bp spacer, the directionality of LoxP sites in the complex is anti-parallel (grey arrows, see (b) for the convention). This orientation is required for correct base-pairing of the 6 bp of exchanged DNA in products. Cre subunits are differentiated into alternating “cleaving” (green) and “non-cleaving” (magenta) conformations, which enforces the pair-wise nature, order and regioselectivity of the exchanges²⁴. The four Cre proteins associate through extensive protein-protein contacts that direct complex assembly. A critical contact involves a domain-swap of the C-terminal helix (CTH) which binds a pocket in the C-terminal domain (CTD) of the adjacent monomer (box).

The two domains of a Cre monomer comprise a clamp that surrounds the 13 bp repeat, creating an extensive interface of protein-DNA contacts²⁴. Monomers are recruited to each LoxP 13 bp repeat and, the active tetrameric recombination complex is subsequently assembled through a pseudo four-fold cyclic arrangement of Cre-Cre interactions (Figure 1c). A crucial inter-subunit contact is a domain swap in which the C-terminal helix (CTH, residues 333–340) packs against the C-terminal domain (CTD, residues 131–326) of an adjacent monomer.

The high fidelity of wildtype Cre for the 13 bp repeat restricts the range of available recombination targets. Efficient reactions are only realized with close matches to LoxP and even single base changes in the 13 bp repeats can nearly abolish efficient function^29–31. This limitation has been circumvented by directed evolution of Cre variants with altered DNA specificity^16,30,32,33. For example, a quintuple mutant CreALSHG (“C2(+/−) #4” from the work of Santoro and Schultz³³), prefers to recombine the LoxP variant, LoxM7. LoxM7 contains three base-pair substitutions at a key protein-DNA interface in the 13 bp repeat (Figure 1b), and is not recombined by wildtype Cre (CreWT).

The homotetrameric Cre-LoxP synapse exerts further substrate restrictions by limiting recombination to occurring between nearly-identical, dyad-symmetric sequences (Figure 2a, i). The symmetric arrangement of protein subunits and their DNA-binding surfaces matches the arrangement of 13 bp repeats of the identical LoxP sites in the complex. As a result, CreWT cannot recombine chimeric Lox sites containing both LoxP and LoxM7 13 bp repeats³⁴ (Figure 1b), since two of the 13 bp repeats do not match the subunit specificities.

Figure 2. Heterotetrameric recombinases.

Complexes are depicted as in Figure 1a, except that the DNA duplexes are represented as a single colored bar. Each color corresponds to a different protein-binding repeat sequence, with corresponding recombinase monomer specificities indicated by the lighter shaded circles. Site directionality is indicated by the pointed end of DNA.

(a) A homotetrameric recombinase (i) will promote recombination between sites with identical or nearly-identical inverted repeats, because the 13 bp repeat arrangement matches the homotetramer pseudo-fourfold symmetry. To allow recombination between dissimilar asymmetric sites, heterotetramers can be created by mixing four different specificity variants (ii), but nearly 70 substrate pairs can be recognized. Mutually-exclusive “orthogonal” interfaces direct heterotetramer assembly to single defined subunit arrangement (iii). This complex will preferentially recombine pairs of sites whose repeats can match this arrangement. For example, an ABCD tetramer can perform ba x dc (left) or bc x da crosses, (right), but not ba x cd crosses.

(b) With two different recombinase specificities, indicated as “CreWT” or “ALSHG” (see Figure 1), 6 unique tetramers are possible (left side). In each, the DNA-binding surfaces match the positioning of one or two of the 10 possible unique substrate pairs arising from different arrangements of two distinct 13 bp repeats (right side). The ABAB tetramer (boxed) should be specific for recombining identical chimeric sites.

Two strategies have been employed to bypass the symmetry restriction of YSSRs. The first strategy utilizes relaxed-specificity recombinases to simultaneously recognize substantially different 13 bp repeats^16,33–35. While this is a convenient approach, such promiscuity could lead to off-target recombination that would be unacceptable for high fidelity applications, such as gene therapy. The second strategy uses combinations of altered-specificity Cre mutants³⁴. A four-variant mixture would permit recombination between two non-identical asymmetric targets with the only requirement being identity in the central 6 bp that are swapped (Figure 2a, ii). However, such a mixture can assemble into seventy unique tetramers, which decreases fidelity by expanding the range of utilizable sequences. Further, currently-available evolved specificity variants may not have high selectivity. For example, CreALSHG achieves 80% of the product levels of CreWT in LoxP integration reactions in vitro and shows only a 2.1-fold affinity preference for LoxM7 (Table 1). Thus, for mixtures, the many combinations of four different moderate-fidelity subunits may lead to partial or full recombination reactions at non-target chromosomal sites. This concern is validated by the cytotoxicity of heterodimeric zinc finger-nuclease chimeras used to introduce programmed double-strand breaks^36,37, which presumably resulted from unintended DNA cleavages from the homodimers.

Table 1.

Complex Assembly and Kinetic Parameters for Cre-Lox Recombination Reactions.^a

		Complex assembly			Substrate turnover
Cre protein(s)	DNA substrates	extentb (%)	S0.5 (nM)	Hill (α)	extentb (%)	A	k1×103 (sec−1)	k2×103 (sec−1)	t1/2 (sec)
CreWT	LoxP x LoxP	47 ± 12	71±5	2.8±0.1	60 ± 6c	0.42±0.02	4.5±1.3	32±3	48
	LoxM7 x LoM7	NRd at 2400 nM complex
CreALSHG	LoxM7 x LoxM7	49 ± 12	150±16	3.7±0.5
	LoxP x LoxP	38 ± 84	313±10	3.6±0.9	20 ± 42	0.66±0.03	1.1±0.2	9.7±0.1	300
CreAAF	LoxP x LoxP	45 ± 44	154±17	2.1±0.2	45 ± 32	0.84±0.01	1.5±0.7	22±4	346
CreWT+	LoxPM7 x LoxPM7	54 ± 54	132±17	3.9±0.7	58 ± 12	0.59±0.07	5.3±0.1	23±1	67
CreALSHG	LoxM7P x LoxPM7	54 ± 22	144±18	4.3±0.4	56 ± 22	0.63±0.03	1.4±0.1	14±2	198
CreAAF+	LoxP x LoxP	21 ± 83	266±22	4.8±2.1
CreALSHG
CreAA+	LoxM7P x LoxM7P	53 ± 55	179±19	3.6±0.6	65 ± 74	0.51±0.13	4.7±0.6	27±6	56
ALSHG-F	LoxM7P x LoxPM7	<3% turnover at 1200 nM complex
	LoxP x LoxM7	9 ± 23	178±9	5.9±1.3	10 ± 82	0.73±0.03	0.93±0.03	33±2	402
	LoxM7 x LoxM7	NR at 360 nM complex
	LoxP x LoxP	reaction in qualitative assay comparable to LoxM7P x LoxM7P
ALSHG-F	LoxM7P x LoxM7P	NR at 360 nM complex
	LoxM7P x LoxPM7	NR at 360 nM complex
	LoxP x LoxM7	NR at 360 nM complex
	LoxM7 x LoxM7	NR at 360 nM complex
	LoxP x LoxP	NR at 360 nM complex
CreAA	LoxM7P x LoxM7P	NR at 360 nM complex
	LoxM7P x LoxPM7	NR at 360 nM complex
	LoxP x LoxM7	NR at 360 nM complex
	LoxM7 x LoxM7	NR at 360 nM complex
	LoxP x LoxP	some products in qualitative assay at > 240 nM complexe

^aComplex assembly and kinetic parameters were calculated as described in METHODS AND MATERIALS, and are given as the averaged values from independent fits of replicate experiments ± S.D. (n > 2) or S.E (n=2).

^bsuperscripts denote number of independent replicates.

^cFrom reference ⁴⁴

^dNR = no detectable reaction in the qualitative assay.

^eFigure 5a (middle panel).

For YSSRs, if a unique heterotetrameric subunit arrangement is enforced, then recombination can be restricted to two pairs of substrates, related by circular permutations of their different recombinase-binding repeats (Figure 2a, iii). Within this pair, the spatial arrangement of repeats would match that of the subunits in the heterotetramer, whereas non-cognate sites in which repeats are not “spatially-matched” would not be efficiently incorporated into recombination synapses. Enforcing assembly of a single arrangement increases the effective site size thereby reducing off-target effects. Applying a similar principle, an engineered heterodimeric intron homing endonuclease preferentially cleaved chimeric DNA substrates over the dyad-symmetric ones with high fidelity³⁸, using a naturally-occurring tandem fusion of the two nuclease DNA-binding domains to assure heterospecificity. However, this strategy cannot be applied to Cre because the N- and C-termini of recombinase monomers are on opposite sides of the recombination complex.

As “proof-of-concept” for the spatial matching principle in Cre-Lox recombination, we directed the assembly of a unique heterotetramer using engineered “orthogonal” heterospecific protein-protein interfaces^39–42 (Figure 3a). We specifically positioned two different Cre monomers in a geometry that promoted efficient recombination between a pair of chimeric Lox sites that spatially-matched the subunit arrangement, while restricting reactivity towards other pairs.

Figure 3. Construction and substrates of the designed Cre heterotetramer.

(a) “Spatial matching” of recombinase and substrates. The alternating ABAB arrangement of CreWT and CreALSHG subunits can be enforced by using two “orthogonal” interfaces surfaces, thereby favoring recombination of substrate pairs containing alternating 13 bp repeat arrangements (left) over un-matched arrangements (middle and right).

(b) “Split interface” variants”. CreAA and ALSHG-F contain complimentary combinations of orthogonal CreAAF and CreWT interfaces with CreWT and CreALSHG DNA specificities. The Ala334-Phe substitution is indicated by the triangular CTH representation and the Met299-Ala/Val304-Ala CTD substitutions are indicated by the wedge-shaped indentation, while the corresponding CreWT interfaces are indicated by the circle and circular indentation.

(c) CTH/CTD Cre-Cre interface. In the CTD (chain B of 1CRX, green), residues Met299, Ala302 and Val304 form a hydrophobic docking surface for residues Ala334, Met335 and Leu338 from the CTH (A chain, purple) from the adjacent subunit. Positions 299, 304 and 334 are the focus of this study and are highlighted in yellow.

(d) Interface residues targeted for engineering. CTD residues Met299 and Val304 form a shallow pocket that accommodates CTH-residue Ala334 from the adjacent subunit (left panel). A hypothetical un-minimized model of the “size-switch” CreAAF variant is shown in the right panel. The Ala299 and Ala304 side-chain truncations form a larger pocket to accommodate the larger Phe334. This volume swap creates alternate interfaces surfaces that interact unfavorably with CreWT surfaces. Contacts between CreAAF and CreWT subunits are disfavored because cavity formation or steric clashes would result.

RESULTS

Heterotetramer design

From the six unique synapses that result from random assortment of two Cre subunits having different DNA specificities, we selectively assembled an ABAB-type heterotetramer (Figure 2b, left). For our two Cre proteins, we chose CreWT and CreALSHG³³ because both are well-characterized using in vitro recombination assays, alone and in combination^34,43. For CreALSHG, the five substitutions confer specificity for LoxM7, mediated structurally by altered direct and water-bridged contacts⁴³. However, it displays significant activity against LoxP, albeit with a 4.4-fold higher S_0.5 value, and a 6-fold slower rate at saturation, compared to CreWT (Table 1).

Two different 13 bp repeats yield ten unique Lox substrate pairs (Figure 2b, right). The ABAB heterotetramer would promote recombination between identical Lox sites containing both 13 bp repeats, but not other substrate combinations. This arrangement requires only two orthogonal Cre-Cre interfaces to impose the alternating subunit positioning (Figure 3a), whereas other arrangements would require four interfaces. Each Cre monomer in the ABAB heterotetramer contains reciprocal orthogonal CTH-CTD combinations to prevent self-oligomerization and enforce heterospecificity (Figure 3b). The CreWT/CreALSHG heterotetramer would be expected to efficiently perform LoxM7P x LoxM7P or LoxPM7 x LoxPM7 reactions but not LoxM7 x LoxP or LoxPM7 x LoxM7P reactions.

Identification of an orthogonal Cre-Cre interface

Heterospecifically-oligomerizing CreWT and CreALSHG subunits were obtained in three steps: 1) isolation of CreWT variants containing alternative functional Cre-Cre interfaces via library screening; 2) identification of one variant, CreAAF, which inefficiently forms synapses with CreWT; and 3) creation of CreWT- and CreALSHG-derived “split-interface” variants, CreAA and ALSHG-F, which contain non-complimenting pairs of wildtype and orthogonal mutant interface surfaces.

To carry of the first step, we introduced amino-acid variation at the CTH-CTD interface, where side chains of CTH residues Ala334, Met335 and Leu338 pack against those of CTD Met299, Ala302, Val304 (Figure 3c). Formation of this contact is required both for tetramer assembly and cleavage activity²⁸. We created an expression library of 8000 residue combinations by specifying all twenty amino acids at three positions that form a contiguous cluster, 299, 304 and 334, in which the large Met and Val side chains pack against the small Ala side chain. The library was transformed into a selection host in which cell survival is promoted by Cre-mediated excision of a conditional-lethal marker. This method was used for both selection and efficiency screening of individual clones (see MATERIALS AND METHODS). From 24 unique sequenced gene variants, five purified candidate proteins were assayed qualitatively for recombination activity in vitro (see MATERIALS AND METHODS). The CreAAF variant contained three substitutions, Met299-Ala, Val304-Ala and Ala334-Phe, and had robust LoxP recombination activity. The pattern of amino acid replacements, with reciprocal large-to-small and small-to-large substitutions (Figure 3d), suggested that CTH-CTD contacts with CreWT would be sterically disfavored. The variant alanine CTD substitutions in conjunction with wildtype Ala334 would be expected to create a destabilizing cavity, whereas the phenylalanine substituted for CTH Ala334 is too large to efficiently against wild type Met299 and Val304 without rearrangements of the CTD interface.

Recombination competences were scored from the dependences of product levels on 2:1 Cre-Lox complex concentrations using a quantitative integration assay^27,31,44, in which Cre-mediated recombination between a synthetic 34 bp Lox site and labeled 220 bp Lox-containing restriction fragment generates 113 bp and 141 bp products (Figure 4a, see MATERIALS AND METHODS). In LoxP x LoxP reactions (Figure 4b), the CreAAF-containing complex yielded a sigmoidal dependence and a saturating level of product formation that were similar to the CreWT complex (44 % vs. 42% turnover), with a two-fold higher S_0.5 value (154 nM vs. 71 nM) (Figure 4c, Table 1). The reduction in apparent affinity could result from smaller contribution of protein-protein interactions to complex assembly equilibria^28,45, suggesting that, although fully functional in terms of product production, the CreAAF CTH-CTD interface is not as avid as in the CreWT complex.

Figure 4. Viability and selectivity of the engineered CreAAF interface.

(a) Cre-Lox recombination assay. 13 bp repeats are indicated in cyan (220 bp) and magenta (34 bp), while the 8 bp spacers are indicated in yellow. Recombination swaps the 13 bp repeats. ³²P-5′-labelled 220 bp Lox-containing restriction fragments (*) were reacted with Cre proteins and 34 bp synthetic Lox (2:1 Cre monomers:Lox site) yielding 141 bp and 113 bp products and HJ intermediates. DNA components were separated by electrophoresis through SDS-PAGE gels, then visualized and quantified by phosphorimaging.

(b) CreAAF is competent for LoxP x LoxP recombination. The concentration-dependences of active complex assembly as indicated by substrate turnover levels were compared for CreWT and CreAAF reactions. The concentrations of 2:1 Cre:LoxP complexes are indicated above each lane. The relevant complexes are diagrammed on the left of the corresponding phosphorimages. CTD and CTH interface surfaces are indicated as described in Figure 3b.

(c) Quantification of LoxP x LoxP titration reactions. Averaged, normalized measurements from 2–4 independent titration experiments and their standard deviations are shown by data points and error bars. The isotherms were generated from averaged fit Hill binding parameters given in Table 1³¹. CreAAF reactions achieved comparable maximal levels to CreWT, but required two-fold higher complex concentrations to achieve 50% maximum turnover.

(d) CreAAF discriminates against wildtype CTH-CTD interfaces. In an “interference” assay, CreAAF and CreALSHG were recruited to adjacent positions on chimeric LoxPM7 sites (bottom left) and their ability to form active complexes was assessed, compared to a heterotetramer containing only wildtype interfaces (CreWT+CreALSHG/LoxPM7, upper left). Reactions were performed as described in (a) and (b).

(e) Quantification of the interference assay. Titration data from (d) were treated as in (c). Data points and error bars depict the normalized averages and their standard deviations for 3 or 4 experiments. The isotherms are calculated from the averaged Hill parameters given in Table 1. CreAAF is less efficient at forming heterodimers with CreALSHG on chimeric LoxPM7 sites compared to CreWT, as evidenced by an increased S_0.5 value and reduced substrate turnover level compared to CreWT+CreALSHG reactions.

We next assayed the exclusion of the CreWT CTH-CTD interface by CreAAF using an in vitro interference assay (Figure 4d). Concentration dependences of substrate turnover in a chimeric substrate recombination reaction, LoxPM7 x LoxPM7, were compared for equimolar mixtures of CreWT+CreALSHG or CreAAF+CreALSHG. Interface incompatibility was indicated by lower efficiency in CreAAF+CreALSHG reactions (Figure 4e). Less substrate was converted to products or HJs (21% vs. 54%), and higher complex concentrations were required to achieve half-maximum turnover (S_0.5 values, 266 nM vs. 132 nM) (Table 1). The lack of complete interference may have resulted, in part, from productive binding to the LoxP 13 bp repeat by a CreALSHG homotetramer. As mentioned earlier, CreALSHG has significant LoxP x LoxP recombination activity, with an S_0.5 value of 313 nM and turning over 38% of substrate at saturation (Table 1). CreALSHG concentration-dependence of recombination activity for chimeric substrates is intermediate between those for LoxP and LoxM7 (data not shown).

Construction and geometric specificity of the engineered ABAB heterotetramer

The CTH and CTD CreAAF substitutions were separated and combined with the complimentary wildtype interfaces (Figure 3b, see MATERIALS AND METHODS for details) to give CreAA, which contains a wildtype CTH with Met299-Ala and Val304-Ala CTD substitutions, and ALSHG-F, which contains a wildtype CTD and Ala334-Phe CTH substitutions. When bound to their symmetric DNA substrates, these “split interface” variants should be disfavored in forming homo-oligomeric contacts. Indeed, ALSHG-F/LoxM7 x LoxM7 reactions showed no LoxM7 recombination product at 540 nM complex, and CreAA required four-fold greater concentrations to achieve CreWT levels of substrate turnover in LoxP x LoxP reactions (Figure 5a). As expected, CreAA or ALSHG-F alone also produced no observable products in either LoxPM7 x LoxPM7 or LoxM7P x LoxM7P reactions, at concentrations up to 360 nM (Figure 5b, left and middle panels). On the other hand, CreAA and ALSHG-F together readily recombined the expected pairs of chimeric substrates, LoxPM7 x LoxPM7 or LoxM7P x LoxM7P (Figure 5b, right panels). Interestingly, the recombination efficiency of the two pairs is not the same, and LoxM7P x LoxM7P reactions achieve higher turnover levels than LoxPM7 x LoxPM7 reactions. In our quantitative analyses below, we used the more efficient LoxM7P x LoxM7P reactions.

Figure 5. Assessment of engineered monomer heterospecificity using a qualitative assay.

(a)–(c) Varying concentrations of 2:1 Cre-Lox complex, indicated above each lane in nanomolar, were reacted with ~ 15 nM supercoiled Lox-containing pLITMUS plasmid for 16 hours⁴⁴ (see MATERIALS AND METHODS), electrophoresed through 1.2 % agarose gels and stained with ethidium bromide. The major products a complex mixture linear monomers and multimers (white arrows, upper left panel), as well as catenanes and plasmid topoisomers. Single site restriction enzyme digests yield the expected pair of product bands and a single reactant band (not shown).

(a) CreAA/LoxP x LoxP (middle) and ALSHG-F/LoxM7 x LoxM7 (right) compared to CreWT/LoxP x LoxP (left) reactions. The lack of complete conversion of supercoiled substrate suggests a greatly reduced recombination and//or topoisomerase activity. ALSHG-F was completely inactive in these assays. CreAA (left) or ALSHG-F (middle) alone exhibited no recombination activity in chimeric substrate (b) LoxM7P x LoxM7P or (c) LoxPM7 x LoxPM7 reactions, but the 1:1 CreAA+ALSHG-F mixture (right) recombined both substrates. However, LoxPM7 x LoxPM7 reactions were much less efficient.

Using the quantitative integration assay, we tested the ability of the CreAA+ALSHG-F heterotetramer to distinguish between chimeric substrates with cognate and non-cognate arrangements of LoxP and LoxM7 13 bp repeats. In LoxM7P x LoxM7P complexes, the cyclic arrangement of repeats spatially-matched the ABAB subunit alternation (Figures 3a and 6a (left)), resulting in efficient LoxM7P x LoxM7P recombination, turning over 54% of substrate with an S_0.5 value of 179 nM (Figures 6a and 6e (upper panel), Table 1). By comparison, a 1:1 mixture of CreWT and ALSHG recombined various chimeric Lox combinations with similar efficiency. For example, in LoxPM7 x LoxPM7 and LoxPM7 x LoxM7P reactions, 54% of substrate is turned over with S_0.5 values of 130-145 nM (Figures 4e, 6d, and 6e (upper panel), Table 1). In the qualitative assay, LoxM7P x LoxM7P and LoxP x LoxM7 reactions behaved similarly (data not shown).

Figure 6. The constrained ABAB heterotetramer distinguishes between different arrangements of LoxP and LoxM7 13 bp repeats.

(a)–(d) Complex structures and electrophoretic analysis of assembly competence and turnover rate. The relevant complexes or substrate pairs are diagrammed on the left. Example phosphorimages are shown of complex assembly titrations (left gel panel) and time course experiments (right gel panel). Titration reactions were carried out as described in Figure 4 and MATERIALS AND METHODS. Complex concentrations in nanomolar are given above the lanes. For time course reactions (1200 nM complex), aliquots were removed at the indicated times, given in seconds, quenched, and electrophoresed. The 220 bp fragment is indicated by the colored dot. The reactions are equimolar CreAA+ALSHG-F with (a) LoxM7P x LoxM7P; (b) LoxP x LoxM7; and (c) LoxM7P x LoxPM7. Similar data for the control reaction of the unconstrained heterotetramer CreWT+CreALSHG in a LoxPM7 x LoxM7P cross are given in (d).

(e) Quantification of titration and time course data. The concentration- (upper panel) or time-dependences (lower panel and inset) of substrate turnover are given for each protein-DNA combination in (a)–(d): CreAA+ALSHG-F/LoxM7P x LoxM7P (filled diamonds, blue line); CreAA+ALSHG-F/LoxPM7 x LoxM7P (filled circles, cyan line); CreAA+ALSHG-F/LoxP x LoxM7 (filled squares, magenta line); and CreWT+ALSHG/LoxPM7 x LoxM7P (open triangles, red line). Average, normalized data points and their standard deviations are indicated, except for CreAA+ALSHG-F/LoxPM7 x LoxM7P, which turned over less than 3% substrate at 1200 nM complex. The curves colors correspond to the reactions indicated by dots at the right of (a)–(d). Complex assembly titration curves were derived from averaged Hill parameters from 2–5 experiments, given in Table 1. The time course progress curves were calculated from a biphasic kinetic model³¹ using averaged parameters from 2–4 experiments, given in Table 1. The inset shows the expanded time course from 0 to 300 seconds.

In contrast to the spatially-matched substrates, the CreAA+ALSHG-F heterotetramer discriminates against LoxP x LoxM7 and LoxM7P x LoxPM7 combinations. These substrate pairs require identical subunits to occupy adjacent positions in the complex (Figures 6b and 6c, (left). LoxPM7 x LoxM7P reactions yielded less than < 3% turnover at up to 1200 nM complex (Figures 6b and 6e (upper panel), Table 1). In LoxP x LoxM7 reactions, more substrate (~9 %) was turned over at saturation, with an S_0.5 value that was similar to the LoxM7P x LoxM7P reactions (Figures 6c and 6e (upper panel), and Table 1). However, 70%, of reacted substrates accumulate as HJ intermediates, compared to 15% for LoxM7P x LoxM7P reactions (data not shown). In other words, CreAA-ALSHG-F exhibits ~17-fold discrimination in product formation between LoxM7P x LoxM7P and LoxP x LoxM7 reactions, similar to the discrimination against LoxPM7 x LoxM7P.

In addition to product yield, the CreAA+ALSHG-F heterotetramer also discriminated kinetically against LoxP x LoxM7 (Figure 6e, lower panel) with a 10-fold lower initial rate compared to LoxM7P x LoxM7P (Table 1). A maximum turnover difference of 12.5-fold is achieved after 3 minutes (Figure 6e, lower panel, inset). The CreAA+ALSHG-F reaction rate was essentially identical to that for CreWT+CreALSHG in LoxPM7 x LoxPM7 reactions (Table 1), but was 2.5-fold faster than the mixture in LoxPM7 x LoxM7P reactions (Table 1, Figure 6e, lower panel).

DISCUSSION

Functionally-useful matches to the large YSSR target sequences are improbable even in large genomes, and the naturally-occurring mouse and human Lox-related sequences do not support efficient recombination by wildtype Cre^32,46. Cre altered-specificity variants can provide access to these, as well as novel symmetric^32,33 or asymmetric sites^16,35. Use of a single relaxed-specificity recombinase to release the symmetry requirement imbues flexibility in target site selection, but such enzymes may be unsuitably promiscuous, leading to DNA cleavage or strand exchange at undesirable sites. Even wildtype Cre has some latitude in the 13 bp repeats it will bind, and reportedly cleaves murine BAC sequences relatively frequently in bacteria⁴⁷. If CreWT can recombine ten different 13 bp variants with reasonable efficiency, which is a somewhat conservative estimate²⁹, then there are ~5000 potential substrate pairs, increasing to ~80,000 if twenty 13 bp repeats are recognized. Heterotetramers can also alleviate symmetry and identity requirements³⁴ but, again, target fidelity is a potential problem. Seventy tetramers, composed of randomly-assorting monomers with different non-overlapping DNA specificities, as estimated above for CreWT, can recognize at least 1.2 million potential substrate pairs. Thus, mixed heterotetramers could exhibit significant off-target reactivity, particularly if the lower fidelity level exhibited by CreALSHG in vitro is typical of evolved recombinase monomers.

Spatially-constrained heterotetramers offer a solution by greatly reducing the number of possible substrates through a specified subunit geometry. For zinc finger-nuclease chimeras, non-target cleavage in vivo was suppressed by enforced heterodimerization using redesigned nuclease dimer interfaces^48,49, indicating that simply limiting homo-dimerization results in useful improvements in target specificity. By comparison, the larger reduction in recombinase oligomerization complexity should effect even greater enhancement.

In this preliminary work, we provide the first evidence that constrained YSSR assembly, using re-configured protein-protein interfaces, can direct recombination to particular asymmetric substrates. The CreAA-ALSHG-F heterotetramer, with its alternating subunit structure, facilely recombined a substrate pair with a cognate alternating arrangement of LoxM7 and LoxP 13 bp repeats, and had low reactivity for arrangements with adjacent identical repeats. In contrast, the freely-associating CreWT+CreALSHG mixture, which can assemble into six distinct tetramers, did not distinguish between these crosses. Together, the combination of binding and kinetic effects yielded an overall discrimination factor at least of 50–170 fold between spatially-matched and unmatched substrates. An unexpected observation was that the CreAA/ALSHG-F heterotetramer had selectivity for LoxM7P x LoxM7P over LoxPM7 x LoxPM7 reactions, with three-fold greater recombination levels for the preferred substrate (unpublished data). This additional specificity level is the topic of a separate investigation, which will be addressed in a later manuscript.

The CreAA-ALSHG-F heterotetramer superficially resembles a natural bipartite recombinase, E. coli XerCD, which recognizes the dissimilar arms of asymmetric dif sites^50,51 with distinct subunits. Like our synthetic analog, XerC or XerD alone exhibit little dif recombination activity⁵², because essential protein-protein interactions coordinate strand exchanges⁵³. The CreAA-ALSHG-F heterotetramer exhibited preferential reactivity, but unlike XerCD, not high fidelity. As predicted, LoxM7P x LoxPM7 recombination levels were very low, perhaps a consequence of assembling the expected heterotetramer in a synapse in which the substrate 8 bp spacers are misaligned in parallel, as opposed to the viable anti-parallel arrangement (Figure 7a, left). Incompatibly-placed asymmetric bends in the 8 bp spacer may preclude stable complex formation²⁸, and the first strand exchange would generate high-energy HJs that would not appreciably accumulate or proceed to the second strand exchange (Figure 7a, middle and right). The low but significant LoxP x LoxM7 reactivity was not predicted, and can be explained by several scenarios. An ABAB heterotetramer would utilize the demonstrated ability of ALSHG-F to react with LoxP repeats, but require CreAA to function on the LoxM7 repeats (Figure 7b, i). Alternatively, homodimer binding of CreAA to LoxP and ALSHG-F to LoxM7 would allow wildtype CTH-CTD interfaces to bridge the two dimer-DNA complexes but would place the non-functional Met299/Val304-Phe334 interface within the ALSHG-F homodimers (Figure 7b, ii). Similarly, a heterotetramer containing three ALSHG-F subunits would place a functional interface on LoxP with promiscuous ALSHG-F occupying a LoxP 13 bp repeat, but at the cost of one bridging interaction (Figure 7, iii). As a consequence, HJ accumulation in this reaction may result from inefficient second exchange by a “mismatched” LoxM7-bound CreAA subunit or an ALSHG-F/ALSHG-F homodimer.

Figure 7. Rationales for CreAA+ALSHG-F reactivities with substrate pairs that are not “spatially-matched”.

(a) Low reactivity of LoxM7P x LoxPM7 recombination. In Cre/LoxP x LoxP recombination complexes, anti-parallel arrangement of LoxP sites insures correct pairing for the central 6 base pairs in the HJ intermediate and product (see Figure 1a). In CreAA+ALSHG-F/LoxM7P x LoxPM7 complexes, the spatially-matched arrangement of 13 bp repeats places the Lox sites in parallel orientation, leading to 4 G-T/AC mismatches in the central 6 base pairs of the HJs and products, adding an 8–10 kcal/mol barrier to strand exchange.

(b) Three possible scenarios for LoxM7 x LoxP recombination. (i) Non-cognate binding of the CreAA-ALSHG-F ABAB tetramer, with un-matched subunits marked with an asterisk (*). While ALSHG has reasonable LoxP recombination activity, CreWT has no measurable activity. (ii) Formation of an A₂B₂ heterotetramer by CreAA and ALSHG-F binding to LoxP and LoxM7 repeats, respectively. While the “bridging” interfaces between dimers are compatible, the intra-dimer interfaces less effective. While CreAA alone can support some LoxP recombination, suggesting that a CreAA/CreAA interface is partially functional while ASLHG-F cannot recombine LoxM7 suggesting that ALSHG-F/ALSHG-F interface is not (Figure 5a). (iii) In an AB₃ CreAA-ALSHG-F heterotetramer, all subunits are bound to 13 bp repeats that support some recombination, but there are only two functional interfaces, one bridging and one intra-dimer.

The LoxP x LoxM7 reactivity suggests that disfavored Cre-DNA or Cre-Cre contacts can be overcome by the cooperative interactions in the complex. Higher-fidelity monomers would partially alleviate this problem. Illustrating the consequence of CreALSHG promiscuity, the CreAA+ALSHG-F heterotetramer performs LoxP x LoxP crosses more efficiently than CreAA alone (data not shown), but does not carry out LoxM7 x LoxM7 crosses, likely because of high CreAA DNA specificity. Greater CreAA-ALSHG-F assembly fidelity would also enhance its substrate bias. For a tetramer, a 0.7 kcal/mol difference at each interface could result in up to 100-fold discrimination in assembly. The selected CreAAF interface exhibited a side chain volume redistribution reminiscent of size-switch combinations in alternate T4 lysozyme cores isolated by similar methods⁵⁴. Such size-swaps afforded 0.9–1.3 kcal increased stability over the cavity-containing or over-packed single mutants, analogous to homodimeric interfaces of CreAA and ALSHG-F, but the apparent discrimination by the CreAAF interface is only an estimated 0.2–0.4 kcal/mol per contact. More exclusive monomer-monomer pairs can be obtained either by increasing the size of the engineered CTH-CTD interface, or by additionally re-engineering N-terminal domain intersubunit contacts.

Homodimer reactivities also suggest different discrimination mechanisms at CreAA/CreAA and ALSHG/ALSHG-F interfaces, with effects on catalysis as well as protein-protein affinity (Figure 5a, middle vs. right panels). The results suggest that the Met299/Val304-Phe334 CTD-CTH combination does not support strand exchange, which is somewhat surprising because cavity-creating substitutions are generally more destabilizing than over-packing ones⁵⁵. The over-packed interface might impede Tyr324 positioning for nucleophilic attack of the scissile phosphate, while the cavity-containing Ala299/Ala304-Ala334 interface may be less avid but also less structurally perturbed.

Besides of the potential for increasing engineered recombinase fidelity, spatially-directed assembly can also be used to investigate subunit interactions within the recombination synapse. Engineered interfaces in conjunction with DNA specificity variants can uniquely place additionally-modified monomers at any of four unique positions, in order to probe the mechanisms and spatial paths of cleavage coordination, assembly cooperativity, and allosteric communication.

MATERIALS AND METHODS

Proteins

The following proteins were used in this study. CreWT: wildtype Cre recombinase with a Met-His₆ N-terminal tag fused to Ser2; CreALSHG: CreWT with five substitutions, Ile174-Ala, Thr258-Leu, Arg259-Ser, Glu262-His, and Glu266-Gly; CreAAF: CreWT with three substitutions, Met299-Ala, Val304-Ala, Ala334-Phe; CreAA: CreWT with two substitutions, Met299-Ala, Val304-Ala; ALSHG-F: CreALSHG with an additional substitution, Ala334-Phe. His-tagged Cre proteins were expressed from pET28b(+)-derived constructs in BL21(DE3) cells (Novagen), purified, and stored as previously described⁵⁶.

DNA

Plasmid DNA encoding CreALSHG was kindly provided by Steve Santoro from the Schultz Lab at TSRI, and the mutations were exchanged into the pET28-His6Cre vector⁴³. Expression vectors for CreAAF and other variants were obtained as described below. Lox-containing pLITMUS plasmids (New England Biolabs), labeled restriction fragments and synthetic Lox DNA substrates were also prepared as described previously⁴⁴. Mutagenic primers were obtained from MWG Biotech (50 nmol scale) and phosphorylated with T4 kinase and ATP. The Lox sites used in this study are shown in Figure 1b.

Heterospecifically-oligomerizing CreAA and ALSHG-F proteins

CreAAF was obtained by in vitro selection for alternate CTD-CTH packing interfaces, which will be described in detail elsewhere. Briefly, a pET28b-His6Cre-based library was constructed in which randomized codons (XXG/C) were substituted for amino acid positions 299, 304, and 334. The library was transformed into an E. coli host harboring a chromosomal rpsL20 streptomycin resistance allele, a DE3 T7 polymerase-expressing prophage (Novagen), and a Cm^r selection plasmid. The selection plasmid contains the wildtype rpsL gene, dominant for streptomycin sensitivity⁵⁷, flanked by LoxP sites in direct orientation. This strain is Cm^r and Str^s. After transformation with the expression library, Cm^r/Str^r/Kan^r cells expressing active Cre variants are selected on triple antibiotic plates, since the dominant Str^s gene is excised via recombinase activity. After DNA extraction and rescreening, 24 unique sequences were recovered from 38 clones. Unique clones were scored for recombination in vivo using the recovery efficiency of the Str^r phenotype. Five candidate proteins, including CreAAF, were purified and tested in vitro for overall activity and oligomerization heterospecificity (see RESULTS).

The heterospecific “split-interface” variants CreAA and ALSHG-F where created by site-directed mutagenesis⁵⁸ of pET28b-His6Cre and pET28b-His6CreALSHG using the following oligonucleotides: Met299-Ala/Val304-Ala, GATCTCCGGTATTGAAgCTCCAGCcCGGGCCgcATCATCTCGCGCGGC and Ala334-Phe, GCGCACCATaaAaCCgGTTTCACT, respectively.

Integrative recombination activity assays

Recombination activity was assessed through Cre-mediated integration of a minimal 34 bp synthetic Lox duplex into a reporter substrate containing a single Lox site in a single turnover reaction ⁴⁴. Reactions are carried out at 21 C in optimized Cre reaction buffer (300 mM lithium acetate, 20 mM Tris-acetate, 1 mM EDTA, 1 mM DTT, pH 8.3). Reactions (50–100 uL total) were initiated by mixing 0.9 volumes reaction buffer and DNA with 0.1 volume of protein mixtures in 300 mM NaCl, 20 mM Tris-acetate, 1 mM EDTA, pH 8.3, quenched with 1 volume of 2x loading buffer (1x = 1% SDS, 20 mM DTT, 6% glycerol, 0.5 mg/mL proteinase K, and 0.05% bromophenol blue) and digested for 1 hour at 37 C prior to electrophoresis. Complex concentrations are expressed as the amounts of 2:1 Cre:Lox (e.g. 1 nM complex = 2 nM total protein + 1 nM synthetic 34 bp Lox site).

For qualitative assessments, the reporter substrate was ~15 nM supercoiled pLITMUS38(+) plasmid containing a 34 bp Lox inserted between the SnaBI and EcoRV sites^43,44. The products were electrophoretically separated using a 1.2% agarose gel in Tris-Acetate-EDTA buffer, and visualized with ethidium bromide under UV light. Qualitative comparisons are made from CCD images of the gels, but rigorous quantitation is unfeasible.

For quantitiative measurements, the reporter substrate was a 220 bp BamHI/StuI restriction fragment from the pLITMUS-Lox plasmid (2 nM or 10 nM), which was 5′-end-labeled with γ³²P ATP and T4 polynucleotide kinase⁴⁴. Samples were quenched and electrophoresed through SDS/10% polyacrylamide gels. A Fuji Image plate was exposed to the dried gels and scanned using Molecular Dynamics Storm 860 image plate reader. Relative labeled DNA band intensities were quantified using ImageQuant. Reaction levels are taken as the amount of substrate that has been converted to 113 bp and 141 bp products and HJ intermediates (“substrate turnover”).

Assessment of complex assembly competence

To assess the strengths of Cre-Cre and Cre-Lox interactions, substrate turnover was measured as a function of complex concentration, by titrating reporter substrate with different Cre-synthetic Lox concentrations in 16-hour endpoint reactions⁴⁴. The complex concentrations typically ranged 15–500 nM (see Figures for amounts). Complex assembly parameters were determined by fitting the amount of substrate turnover (v′) and total complex concentration ([complex]) to the function v′ = (f·[complex]^α/(S_0.5^α + [complex]^α)⁴⁴. The fit parameters were f, the maximum amount of substrate turnover; S_0.5, the complex concentration when half the maximum product is produced (an apparent dissociation constant); and α, the apparent Hill coefficient (reported as the average of two to five independent experiments ± the standard deviation in Table 1). The S_0.5 value is a complex function of monomer DNA binding, dimerization and tetramer assembly equilibria. The apparent α value likely reflects, in part, the degree of cooperativity arising from protein-protein interactions in complex assembly.

Single turnover kinetics

In time course reactions, 10 nM ³²P-labeled reporter was reacted with saturating complex (2400 nM Cre and 1200 nM Lox). Reactions were initiated by addition of complex and quenched after 30 to 1920 seconds. Rate parameters were determined by fitting the measured percent substrate reacted (v′) and reaction time (t) to the function v′ (t) = f·(1 − [Ae^−k1t + (1 − A)e^−k2t]). The fit parameters were f, the percent of substrate turnover at t = ∞; A and k₁, the amplitude and rate constant for the reaction slow phase; k₂, the rate constant for the fast phase (reported as the average of two to four independent experiments ± the standard deviation in Table 1). The biphasic function is usually required to adequately fit the data⁴⁴, suggesting that distinct fast- and slow-reacting complexes are formed early in the assembly process, but the data do not distinguish between a converging or sequential relationship between the two paths.

ABBREVIATIONS USED

YSSR

tyrosine site-specific recombinase

CreWT

wildtype Cre recombinase with a Met-His₆ N-terminal tag fused to Ser2

CreALSHG

CreWT with five substitutions, Ile174-Ala, Thr258-Leu, Arg259-Ser, Glu262-His and Glu266-Gly

CreAAF

CreWT with three substitutions, Met299-Ala, Val304-Ala and Ala334-Phe

CreAA

CreWT split interface mutant with two substitutions, Met299-Ala and Val304-Ala

ALSHG-F

CreALSHG split interface mutant with an additional substitution, Ala334-Phe

LoxP

natural Cre recombinase recognition site

LoxM7

LoxP variant that is the preferred substrate of CreALSHG, containing T7-C, C8-T and G9-A left arm 13 bp repeat and the dyad-related C26-T, G27-A and A28-G right arm 13 bp repeat substitutions

LoxPM7

chimeric Lox site with a LoxP left arm 13 bp repeat and a LoxM7 right arm 13 bp repeat

LoxM7P

chimeric Lox site with a LoxM7 left arm 13 bp repeat and a LoxP right arm 13 bp repeat