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The λ Integrase Site-specific Recombination Pathway

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  • Author: Arthur Landy1
  • Editors: Phoebe Rice2, Nancy Craig3
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Division of Biology and Medicine, Brown University, Providence, RI; 2: University of Chicago, Chicago, IL; 3: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0051-2014
  • Received 24 August 2014 Accepted 11 November 2014 Published 26 March 2015
  • Arthur Landy, arthur_landy@brown.edu
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  • Abstract:

    The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in , there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics.

  • Citation: Landy A. 2015. The λ Integrase Site-specific Recombination Pathway. Microbiol Spectrum 3(2):MDNA3-0051-2014. doi:10.1128/microbiolspec.MDNA3-0051-2014.

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/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0051-2014
2015-03-26
2017-08-20

Abstract:

The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in , there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics.

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FIGURE 1

λ Integrase and the overlapping ensembles of protein binding sites that comprise site DNA. The left panel shows the structure of a single λ Int protomer bound via its NTD to an arm site DNA and via its CTD to a core site DNA (adapted from the Int tetrameric structure determined by Biswas . [ 44 ], PDB code 1Z1G). The right panel shows the recombination reactions. Integrative recombination between supercoiled P and linear B requires the virally encoded integrase (Int) ( 2 ) and the host-encoded accessory DNA bending protein integration host factor (IHF) ( 4 , 177 ) and gives rise to an integrated phage chromosome bounded by L and R. Excisive recombination between L and R to regenerate P and B additionally requires the phage-encoded Xis protein (which inhibits integrative recombination) ( 140 ) and is stimulated by the host-encoded Fis protein ( 8 ). Both reactions proceed through a Holliday junction intermediate that is first generated and then resolved by single strand exchanges on the left and right side of the 7 bp overlap region, respectively. The two reactions proceed with the same order of sequential strand exchanges (not the reverse order) and use different subsets of protein binding sites in the P and P′ arms, as indicated by the filled boxes: Int arm-type P1, P2, P′1, P′2, and P′3 (green); integration host factor (IHF), H1, H2, and H′ (gray); Xis, X1, X1.5, and X2 (gold); and Fis (pink). The four core-type Int binding sites, C, C′, B, and B′ (blue boxes) are each bound in a C-clamp fashion by the CB and CAT domains, referred to here as the CTD. This is where Int executes isoenergetic DNA strand cleavages and ligations via a high-energy covalent 3′-phospho-tyrosine intermediate. The CTD of Int and the tetrameric Int complex surrounding the two overlap regions are functionally and structurally similar to the Cre, Flp, and XerC/D proteins. Reprinted with permission from reference 36 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f1

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FIGURE 2

Formation, resolution, and trapping of Holliday junctions (HJ). (A) The top strand of each att site is cleaved via formation of a high-energy phosphotyrosine intermediate and the strands are exchanged (three bases are “swapped”) to form the HJ, thus, creating a branch point close to the center of the overlap regions. A conformational change of the complex that slightly repositions the branch point and more extremely repositions the Int protomers leads to the second swap of DNA strands and resolution of the HJ to helical products ( 44 , 178 ). These features of the reaction suggested the mechanism-based method of trapping HJ complexes shown in (B). The left panel shows the DNA sequence changes made in the 7 bp overlap regions to trap HJ intermediates (lower case letters). Following the first pair of Int cleavages (via the active site Tyr) on one side of the overlap regions (arranged here in antiparallel orientation), the “top” strands are swapped to form the HJ; this simultaneously converts the unpaired (bubble) bases to duplex DNA. On the other side, the sequence differences between the two overlap regions strongly disfavor the second (“bottom”) strand swap that would resolve the HJ, because this would generate unpaired bubbles in the product complex ( 36 , 37 , 38 , 39 ). This diagram applies to both integrative and excisive recombination (even though the labels refer to integrative recombination). Adapted in part, with permission, from reference 36 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f2

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FIGURE 3

X-ray crystal structure of the Int CTD. (A) With this modified version of previously designed suicide recombination substrates ( 35 , 47 ) covalently trapped CTD-DNA complexes were stable for weeks. Formation of the phosphotyrosine bond and diffusion of the three base oligonucleotide is followed by annealing of the three base flap to the three nucleotide gap, thus, positioning the 5′-phosphate such that it repels water and shields the phosphotyrosine linkage from hydrolysis. (B) Ribbon diagrams showing the central domain (residues 75 to 160; above the DNA) and the catalytic domain (residues 170 to 356; below the DNA) of λ Int, and their interactions with the major and minor grooves on the opposite sides of the DNA. A long, extended linker (residues I160 to R176) connects these domains. The scissile phosphate that is covalently linked to Y342 is shown as a red sphere. The central domain inserts into the major groove adjacent to the site of DNA cleavage. The catalytic domain makes interactions with the major and minor groove on the opposite side of the DNA, straddling the site of DNA cleavage. (C) The solvent accessible surface of the Int protein is shown, colored according to electrostatic potential. The DNA binding surface is highly positive (blue) and makes numerous interactions with the phosphates of the DNA (cf. Figure 3B ). The polypeptide linker between domains joins the central and catalytic domains on one side of the DNA. A salt bridge between the Nζ of K93 and the carbonyl oxygen of S234 bridges between domains on the other side of the DNA, completing the ring-shaped structure that encircles the DNA. (D) The architecture of the λ Int C-75 protein is shown with cylinders and arrows representing helices and β strands, respectively. This view is oriented similarly to that in (A) (right side). The central domain of λ Int lacks helix E, corresponding to the fifth helix of Cre's N-terminal domain, which is involved in subunit interactions. Reprinted with permission from reference 45 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f3

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FIGURE 4

A Remodeling of Int's active site switches DNA cleavage activity on and off. (A) A comparison of the DNA-bound (left), and unbound (right), structures of λ Int shows a dramatic reorganization of the C-terminal region spanning residues 331 to 356 (red). In the absence of DNA, Y342 (yellow) is far from the catalytic triad of R212, H308, and R311 (magenta side chains). In the DNA complex (left panel), Y342 has moved into the active site. Another consequence of the DNA-bound conformation is that the extreme C-terminal residues 349 to 356 extend away from the parent Int molecule and pack against another molecule in . (B) A cartoon illustrating how the DNA-bound conformation of Int positions the Y342 for cleavage of DNA. The isomerization from the inactive form, in which Y342 is held some distance from the catalytically important Arg212-His308-Arg311 triad ( 65 ), to the active conformation seen in complex with DNA, is accompanied by the release of strand β7 and its repacking in against a neighboring molecule. (C) The assembly of active (orange) and inactive (gray) catalytic sites results from a skewed packing arrangement of λ Int subunits (residues 75 to 356) in the tetramer. The scissile phosphates bound by active and inactive subunits are shown as red and gray spheres, respectively. Reprinted with permission from references 44 and 45 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f4

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FIGURE 5

Structure of the λ Int tetramer bound to a Holliday junction and arm DNAs. (A) The domains of Int pack together as three stacked layers, with the NTDs cyclically swapped onto neighboring subunits. The NTD layer embraced by two antiparallel arm DNAs is linked through short α-helical couplers to the CTD, which encircles the branches of the Holliday junction. The active subunits are colored red/green and the inactive subunits are blue/yellow. (B) The 2-fold symmetry of the NTD layer is reflected in the skewed arrangement of the CTDs and the shape of the four-way junction (thick dark gray lines) in the bottom strands reactive isomer. Reprinted with permission from reference 44 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f5

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FIGURE 6

Three different conformations of λ Int tetramers representing distinct steps of the recombination reaction. The core DNAs within the λ-Int synaptic complex (A, D), the λ-Int post- strand exchange complex (B, E), and the λ-Int Holliday junction complex (C, F) are shown along with schematic diagrams illustrating the interbranch angles and position of branch points. The pair of Int subunits in the active conformation (orange/red) is positioned closer to the center of each complex, whereas the inactive pair of subunits (gray) is further apart. Scissile phosphates (spheres) activated for cleavage are colored in red. Reprinted with permission from reference 44 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f6

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FIGURE 7

Complex of integration host factor with H′1N. The α and β subunits are shown in white and pink, respectively. The consensus sequence is highlighted in green and interacts mainly with the arm of α and the body of β. The yellow proline at the tip of each arm (P65 α/P64 β) is intercalated between bp 28 and 29 on the left side and 37 and 38 on the right. Reprinted with permission from reference 4 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f7

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FIGURE 8

Complex of Xis with DNA. (A) The structure of Xis specifically bound to X2 DNA penetrates adjacent grooves of the duplex by fastening on the phosphodiester backbone. The major groove is filled primarily with helix α2 with the side chains of Glu19, Arg23, and Arg26 playing a major role in specific DNA recognition. The adjacent minor groove is contacted by the “wing” which does not contribute significantly to the specificity of complex formation but does contribute to binding affinity, although to a smaller extent than helix α2. The side-chain of Arg39 (brown) extends along the floor of the minor groove where it makes direct and water-mediated hydrogen bonds. (B) A model for the Int (NTD)-Xis-DNA ternary complex. The Int (NTD) is modeled to interact with the TGA trinucleotide (underlined) of the P2 site (blue) in the DNA major groove. Xis is modeled on the X1 site (magenta) in the same manner as observed in the complex with the X2 site. The C-terminal tail of Xis, which is disordered in solution (not shown), is located adjacent to the C-terminal helix of the NTD of Int to make a protein–protein interaction as shown by mutagenesis and NMR titration data ( 179 ). (C) X-ray crystal structure of Xis bound to the Xis binding region reveals the structural basis of cooperative binding. Xis monomers bound to the X1, X1.5, and X2 sites are colored dark salmon, green, and blue, respectively. (D) Structure-based model of an extended Xis-DNA filament. Units of the Xis-DNA crystal structure were stacked end-to-end by superimposing site X1 over X1.5 to assemble a pseudocontinuous helix with a pitch of ∼22 nm. Proteins are blue; DNA is orange. Reprinted with permission from reference 139 (A and B) and reference 140 (C and D). doi:10.1128/microbiolspec.MDNA3-0051-2014.f8

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FIGURE 9

X-ray crystal structure of a Fis dimer complexed with DNA (A) and its relation to Xis binding (B). (A) The C-terminal helix representing the recognition helix of the HTH unit of each subunit is inserted into adjacent major grooves on the concave side of the 21 bp curved DNA. Only base contacts with a single residue, Arg85, are important for binding. The DNA undergoes substantial conformational adjustments, including adoption of ∼65° overall curvature, to fit onto the Fis binding surface. The central 5 bp of the DNA interface are not contacted by Fis, but compression of the central minor groove to almost half the width of canonical DNA at the center enables the α-helices to insert into the adjacent major grooves, which do not show any appreciable change in width. (B) Model of the Fis-Xis cooperative complex. The X-ray crystal structure of three Xis monomers bound to the X1 (magenta), X1.5 (blue), and X2 (gold) binding sites was superimposed onto the model of the Fis K36E X-ray structure docked to DNA representing the F site. Fis subunits are cyan and yellow. The DNA recognition helices of Xis bound at X2 and the proximal Fis subunit nearly form a continuous protein surface within the major groove. Reprinted with permission from reference 7 (A) and reference 8 (B). doi:10.1128/microbiolspec.MDNA3-0051-2014.f9

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FIGURE 10

Schematic summary of the Int bridges in integrative and excisive recombination. The middle panel diagrams the Int bridges of the Holliday junction (HJ) recombination intermediates determined by Tong . ( 36 ). In the integrative complex, all four core sites and four of the five arm sites enjoy an Int bridge while the excisive complex engages three of the four core sites and three of the five arm sites. The flanking panels (brackets) depict extrapolations from the HJ complexes to the respective site recombination partners (substrates) and recombinants (products) based on the deduction that Int bridges are not broken and reformed during recombination. doi:10.1128/microbiolspec.MDNA3-0051-2014.f10

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FIGURE 11

Models of the λ excisive and integrative recombination complexes. (A) Schematic representation of the excisive complex architecture. The excision reaction product resulting from Holliday junction (HJ) resolution is shown. Int subunits (blue, green, magenta, brown) are represented by a small circle (NTD) and a large circle (CTD). Integration host factor (IHF) heterodimers (gray) are shown bound to the H′ and H2 sites. Fis dimer (pink) and Xis (tan) subunits are indicated. (B) Model of the excisive complex in the same “top view” orientation as the schematic drawing in panel A. The NTD of the Int subunit bound at the C core site (NTD-C) is shown separated from the rest of the complex to improve clarity of the P-arm trajectory. (C) Side view of the excisive complex, highlighting the trajectory of the P-arm. IHF bending of the P′ arm at H′ directs the DNA over the CTD domains of the Int tetramer, facilitating engagement of the P′1 and P′2 arm sites by the Int subunits bound at the C′ and B core sites, respectively. In the P-arm of R the phasing of the IHF-induced bend at H2 is different from that at H′; at H2, the P-arm is directed along the plane of the catalytic domain tetramer. An A-tract sequence that is stabilized by Fis binding ( 7 , 8 ) directs the P-arm upwards, towards the Int CB domains. The cooperative Xis filament ( 8 , 140 ) then redirects the P-arm across the top of the Int CTD domains, where the P2 site is bound by the Int subunit bound at the B′ core site. The Xis subunit bound at X1 resides close to the position where the NTD of the Int subunit bound at the C core site (Int-C) would be expected. The NTD of Int-C was not docked in a specific location of the excisive complex model, but it seems plausible, even attractive, that this domain could bind nonspecifically to the P-arm near the X1 site, perhaps interacting with Xis. (D) Schematic of the integrative complex architecture. The arm-type binding sites engaged by the four Int subunits are indicated. (E) Model of the integrative complex in the same “top view” as illustrated in panel B. In this orientation, the P-arm rises towards the viewer, crosses over the P′ arm, and is directed back towards the Int tetramer by the IHF bend at the H1 site. (F) Side view of the integrative model, looking approximately down the B core site. The NTD of the Int subunit bound at the B core site (NTD-B) is shown bound at the P1 site, on the flexible P-arm. The CB and catalytic domains of the Int subunit bound at the B site can be seen wrapped around the opposing face of B, with the interdomain hinge indicated. The CTD-NTD linkers were not modeled and are not shown. IHF bending at H′ directs the P′ arm over the CTD domains of the Int tetramer, but in this case the P′1, P′2, and P′3 binding sites are engaged by the Int subunits bound to C′, C, and B′, respectively. As Xis is not present in the integrative complex, the P-arm is directed upwards, parallel to the Int tetramer, and as Fis stimulation of integration has been reported ( 180 , 181 ), it was included in the model. IHF bound to the H1 site redirects the P-arm back towards the Int tetramer, crossing over the P′ arm in the process. The P1 arm-type site is thereby brought to a position where it can bind the NTD of the Int subunit poised for capture of the B core half-site (Int-B). Reprinted with permission from reference 37 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f11

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0051-2014
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FIGURE 12

Schematic representation of the excisive and integrative reactions, based on the structural models shown in Fig. 13 . Coloring of the protein subunits matches that shown in Fig. 11 . Reprinted with permission from reference 37 . doi:10.1128/microbiolspec.MDNA3-0051-2014.f12

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0051-2014
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FIGURE 13

The basis for directionality in λ recombination. (A) An explanation for why Xis is required for excision. (B) Explanation for why the excision pathway is not run efficiently in reverse to perform integration. (C) Explanation for why Xis inhibits the normal integration reaction. The Xis, P2, and H1 sites cannot be occupied simultaneously ( 22 ). Schematics follow the same coloring scheme used in Fig. 11 and Fig. 12 . Int subunits not bound to a core site are colored gray. doi:10.1128/microbiolspec.MDNA3-0051-2014.f13

Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0051-2014
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