Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase

Bai, Hua; Sun, Mingxuan; Ghosh, Phalguni; Hatfull, Graham F.; Grindley, Nigel D. F.; Marko, John F.

doi:10.1073/pnas.1018436108

Cited by 63 publications

(84 citation statements)

References 25 publications

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“…A recent single-molecule and topological analysis of the reactions catalyzed by the serine integrase of mycobacteriophage Bxb1 provided evidence of unconstrained rotation consistent with the first scenario (18). It was concluded that the cleaved intermediates remained "rotationally open" for periods of up to several minutes, allowing equilibrium between negative supercoiling and topological entanglement to be reached.…”

Section: Discussionmentioning

confidence: 83%

“…Experimental analysis of iteration product topologies provided crucial evidence in support of subunit rotation by small serine recombinases (9,(13)(14)(15)(16)(17). Recently, data consistent with extensive repeated rounds of subunit rotation by the serine integrase Bxb1 were obtained by single-molecule and gel electrophoretic analysis (18). It was concluded that the synaptic intermediate containing two cleaved att sites and Bxb1 integrase can remain rotationally open for many minutes, allowing many rounds of rotation to take place.…”

mentioning

confidence: 90%

“…This situation occurs when the overlap sequences of the two sites are different, or when sites with the same, asymmetric overlap sequence are aligned in an antiparallel way (see above). Recombinant products are usually not observed; instead, a second round of strand exchange may restore the nonrecombinant configuration, allowing religation but altering the DNA topology (14,15,18,19,22,23) (Fig. 3A).…”

Section: Topology Of Multiple Rounds Of Strand Exchange In Mismatchedmentioning

confidence: 99%

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Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Olorunniji

Buck

Colloms

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180°rotation. However, multiple processive "360°rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.T he large serine recombinase ϕC31 integrase (605 amino acids) promotes integration of the bacteriophage ϕC31 genome into the Streptomyces host chromosome by recombination between a phage site attP and a bacterial site attB, resulting in an integrated prophage flanked by two recombinant sites, attL and attR (1, 2). The prophage can remain dormant for many generations in this lysogenic state until it is excised by integrasemediated recombination between attL and attR, reforming attP and attB sites. The att sites (∼50 bp) each comprise sequences recognized by integrase flanking a central 2-bp overlap sequence at which crossover occurs (3-5) ( Fig. 1C and Fig. S1). Two integrase subunits bind to each att site, forming on-site dimers. Two att sites that are to recombine are then brought together by dimer-dimer interactions. ϕC31 integrase and related serine integrases catalyze efficient recombination between attP and attB, but not between the prophage sites attL and attR, or any other pairs of sites. However, in the presence of a phage-encoded recombination directionality factor, integrase specificity is altered; attL × attR recombination is efficient and attP × attB recombination is inhibited (4, 6, 7). There is considerable interest in this group of recombinases as tools for applications in biotechnology and genetic manipulation (8).Previous studies on the mechanism of recombination by small serine recombinases have led to a "subunit rotation" model for strand exchange (9-11). In this model, cleavage of all four DNA strands in a synaptic complex of the two recombining sites cre...

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Section: Discussionmentioning

confidence: 83%

mentioning

confidence: 90%

Section: Topology Of Multiple Rounds Of Strand Exchange In Mismatchedmentioning

confidence: 99%

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Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Olorunniji

Buck

Colloms

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…As demonstrated for other SRs, DNA exchange is believed to be mediated by double-strand DNA cleavages at the centers of each att site, translocation of one synaptic pair of subunits relative to the other within the tetramer, and religation of the four DNA strands (30)(31)(32)(33). The chemical and protein isomerization events largely involve the N-terminal catalytic domain and long oligomerization helix in each subunit, which bear clear similarities to well-studied members of the small SR family.…”

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confidence: 99%

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

et al. 2017

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The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.KEYWORDS site-specific DNA recombination, serine integrase, recombination directionality factor, Listeria monocytogenes T emperate phage genomes integrate into and excise out of bacterial chromosomes using phage-specific site-specific recombination systems. Many phage integrases are members of a subfamily of serine recombinases (SRs) that are known as large SRs due to their very long C-terminal DNA binding regions relative to other SRs (1-4). In the few serine integrase systems that have been studied, phage-encoded recombination

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“…To overcome complications caused by ensemble averaging and observe site-specific recombination in real time, various single-molecule techniques have been used, including atomic force microscopy (11), tethered particle motion (TPM) (4,12), and magnetic tweezers (13). In particular, the recent TPM work by Fan et al (12) observed the overall progress of recombination reactions, measuring various association and dissociation rates as well as noting the heterogeneous behavior of complexes, but was unable to correlate these changes to nanometer-scale rearrangements within individual complexes.…”

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confidence: 99%

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Pinkney

Zawadzki

Mazuryk

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large-and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.tethered fluorophore motion | site-specific DNA recombination | Holliday junction dynamics | alternating laser excitation | protein-DNA interactions S ite-specific DNA recombination is the protein-mediated cleavage, exchange, and rejoining of DNA strands between two duplexes containing a specific sequence. Tyrosine recombinases are involved in the integration and excision of viral genomes, resolution of chromosome dimers, and gene expression (1). This large family of proteins shares a general mechanism of recombination whereby a tetramer of recombinases mediates sequential pairs of strand exchange between two DNA duplexes; the first pair forms a Holliday junction (HJ) intermediate, whereas the second leads to resolution of the HJ to recombinant product (Fig. 1A). DNA cleavage requires no external energy factors, as the bond energy is stored during strand exchange as a covalent protein-DNA linkage.One of the best studied tyrosine recombinases is Cre, a 38 kDa protein of the bacteriophage P1 of Escherichia coli. Cre catalyzes cyclization of the viral genome and resolution of plasmid dimers during replication (2). Cre mediates recombination between 34 bp DNA sequences named loxP (Fig. 1B). Unlike other recombinases [e.g., XerCD (3) and λ Int (4)], Cre does not require accessory...

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Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase

Cited by 63 publications

References 25 publications

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

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