Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination

Abstract

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Keywords: Listeria monocytogenes; phage A118; serine recombinase; site-specific DNA recombination; synaptic complex.

FIG 1

The A118 integrase reactions and structural models. (A) Phage A118 integration and excision. The A118 integrase recombines the attP site on the phage genome (thick line) with the attB site in the comK gene of the Listeria chromosome (thin line) in a reaction inhibited by the phage-encoded Gp44 protein. Gp44 is required for the integrase-catalyzed excision of the prophage between the hybrid sites attL and attR to regenerate the circular extrachromosomal phage genome. (B) Schematic representation of the domains of the integrase dimer bound to the different att site sequences. The catalytic domain (cat) is connected to the recombinase domain (RD) on the opposite side of the DNA duplex through the helix E region (in brown). The zinc-containing domain (ZD), which is connected to the RD via a flexible peptide linker, can be positioned on either side of the DNA helix depending on the location of its DNA recognition sequence. DNA exchange occurs over the red GG core dinucleotide. The color-coding of the Int domains and CC motifs (red) is maintained throughout the figure. (C) Structural models of the integrase dimer bound to attP, attB, and attL. The mobile CC motifs (red) are oriented in these models as in chain C in the structure under PDB accession number 4KIS, except for the CC-paired conformation at attL. In the attL CC-paired model, the CC dimer structure, based on the structures under PDB accession numbers 5U96 for chains A and B and 4KIS for chain C, was manually positioned onto the Int-attL model. No connections were made over the unstructured peptide segments between the ZDs and the CC motifs (see also reference 31). The helix E segments are yellow in this panel to better visualize the paired CC motifs. (D) Schematic models of attB × attP and attL × attR synaptic complexes. Modeling suggests that considerable flexibility within the CC motifs is required to achieve interactions between the tips of the CC motifs of interacting integrase dimers. Gp44 (tan) binding to the base of the CC motif and ZD is proposed to be required to reorient the CC motifs in attL and attR from the paired configuration within dimers (C, bottom) to trajectories competent for interaction between dimers to achieve attL × attR synapsis (30).

FIG 2

Reconstructed att sites. (A) DNA sequences of the native attP, attB, attL, and attR sites and those reconstructed from attP and attB by changing the spacing between the ZD and RD recognition determinants. Red bases are the core nucleotides, and base identities between attP and attB when aligned as shown are marked. (B to E, left) Gels representing products of 20-min Int reactions between 100-bp DNA fragments containing wt and reconstructed att sites and supercoiled (sc) plasmids containing attR (B), attL (C), attB (D), and attP (E). oc is open-circular plasmid. Reaction mixtures for panels B and C included Gp44. The recombinant product (rec) is a linear plasmid (Fig. 1A). (F) Summary table of the recombination specificities of wt and reconstructed att sites.

FIG 3

Electrophoretic mobilities of Int and Int+Gp44 bound to reconstructed att sites. The binding of the Int dimer shifts the mobilities of the DNA fragments. Gp44 binding further retards Int complexes on attP, attL, and attR but increases the electrophoretic mobility on attB complexes, even though the mass is increased by 17 kDa. The differences in migrations are believed to reflect the architectures of the different complexes, specifically the trajectories of the CC motifs.

FIG 4

The length of the CC motif is important for activity. (A) Alignment of the amino acid residue sequences of the CC segments from the A118 and TP901 integrases. Below are the sequences of the CC length mutants. (B) Structure of the L1 integrase RD (green) and ZD (blue) rendered as a transparent surface. The CC motif (PDB accession number 4KIS for chain C with the tip appended from PDB accession number 5U96 for chain A) is highlighted in red; cyan denotes the segment deleted in A118 CCΔ7 or replaced with TP901 residues (A118 chimera). (C) attP × attB deletion reactions by the wt and CC mutant integrases. On the left are plots of representative reaction time courses, and on the right are gel images of reactions by the Int CC chimera and Int CC+7 mutants. The DNA was digested with NdeI, which cleaves the parental (par) plasmid twice and linearizes the product circles, prior to agarose gel electrophoresis (see Fig. S4 in the supplemental material). (D) Gp44 inhibition of attP × attB deletion reactions by wt and mutant integrases. Reactions were performed as described above for panel C for 20 min in the presence of 0, 25, 50, 100, 150, and 200 nM Gp44, and example data are shown. (E) Gp44-activated attL × attR deletion reactions by the wt and CC mutant integrases. Reactions were performed as described above for panel D.

FIG 5

Alanine-scanning mutagenesis of residues over the tip of the CC motif. (A) Structure of CC pairs over their tip regions from the structure under PDB accession number 5U96. On the left, the AB and GH pairs were aligned over chain A (red) and chain G (pink) to highlight the rotational differences between interacting chain B (silver) and chain H (green), respectively. On the right are the AB and GH CC pairs with selected interacting side chains shown to illustrate differences in the interfaces. The ion pair between Lys362 and Glu378 is denoted on CC pair AB, but it is not present in CC pair GH. See Fig. S1 in the supplemental material for a rotated (end-on) view of the interface highlighting differences. (B) Representative intramolecular attP × attB deletion reactions by a set of CC alanine mutants. (C) Representative intramolecular Gp44-activated attL × attR deletion reactions by the same set of CC alanine mutants. Reactions in panels B and C were done for 20 min. (D) Bar graph depicting the relative reaction efficiencies for attP × attB and attL × attR reactions by each mutant. Percent recombination values from 2 to 4 deletion reactions (20 min) were averaged and scaled relative to Int-wt. (E) Representative time courses of intermolecular fusion reactions by Int-wt and selected mutants showing very low recombination rates for both attP × attB (left) and attL × attR (right) (Int-L379A) or poor recombination for attP × attB in comparison to attL × attR (Int-L368A and -Y374A). (F) Representative time courses of attP × attB and attL × attR intermolecular reactions by Int-wt and Int-D367A, which is more defective for attL × attR reactions.

FIG 6

Electrophoretic migrations of CC alanine mutants bound at attP and attL. Int CC mutants were bound to 100-bp DNA fragments containing attP (A) or attL (B) and subjected to native PAGE (37.5:1 polyacrylamide-bisacrylamide) (Fig. S2 in the supplemental material shows examples at a 59:1 acrylamide ratio). Slow migrations by CC mutants relative to Int-wt observed on attL correlate with defective recombination and imply a less compact structure that is consistent with the CC motifs in a more open configuration. The binding of Gp44 to mutant Int-attL complexes abrogates the mobility differences (Fig. S3).

FIG 7

Helix E residues controlling synaptic complex formation. (A) Sequence of the N-terminal segment of the E helices of SRs over the region in contact with their catalytic domain or partner helix E. Blue-shaded residues are the positions of gain-of-function mutations in A118 integrase (this work), Hin DNA invertase (38, 39, 43), Tn3 or γδ resolvase (37, 41), Sin resolvase (42), and ϕC31 Int (61) that enhance SC formation. Three LSR sequences are also given with gray shading, indicating identities with the A118 integrase: a putative conjugative transposon recombinase from Clostridium difficile used for structural modeling (PDB accession number 3G13) (see panel H), the TP901 integrase, and the more distantly related ϕC31 integrase. (B) SC formation by A118 mutants. Reactions with the different A118 proteins employed 100-bp ³²P-labeled attB and excess 60-bp attP fragments. Proteins in the right panel have their CC motifs deleted. (C) Migration of SCs formed by Int-M117I and -L127V with 100-bp ³²P-labeled attB and excess 60- or 200-bp attP fragments. (D) Specificity of att site synapsis by A118 mutants. Example gels are given for Int-M117I and -L121I reactions with 100-bp ³²P-labeled attB and excess 60-bp attB, attP, attL, and attR fragments in the absence and presence of Gp44. Note that Gp44 binding (*) to the Int dimer on attB shifts the complex to a faster-migrating species. A complete set of specificity gels is provided in Fig. S5 in the supplemental material. (E) Summary of att site synapsis specificities by the A118 integrase mutants. +^f indicates a higher-mobility complex that is missing the attP DNA. (−) indicates not detected but that a higher-mobility SC could be obscured by the Gp44-bound dimeric complex (but see Fig. S5B). (F) SEC of Int-M117 without and with incubation with attP DNA. The trace of the DNA only (60-bp attP) and peaks of MW standards (catalase, 232 kDa; aldolase, 158 kDa; BSA, 67 kDa) are shown. AU, arbitrary units. (G) Recombination frequencies of wt and mutant A118 integrases with or without their CC motifs. Results are the averages and standard deviations from 20-min attB × attP deletion reactions relative to the wt (0.556 ± 0.033 deletions/substrate molecule). (H) Structural model of the A118 integrase dimer over the catalytic domain and helix E regions highlighting the locations of the SC-forming mutations in helix E. The catalytic domain of one subunit is rendered as a surface with its helix E as a dark green ribbon and the native side chains of the mutant residues in red (C_α red sphere for Gly119). The model is based on the structure under PDB accession number 3G13 (see Materials and Methods).