The mechanism of ϕC31 integrase directionality: experimental analysis and computational modelling

Pokhilko, Alexandra; Zhao, Jia; Ebenhöh, Oliver; Smith, Margaret C. M.; Stark, W. Marshall; Colloms, Sean D.

doi:10.1093/nar/gkw616

Cited by 21 publications

(56 citation statements)

References 33 publications

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“…An alternative explanation is that the plateau behavior reflects an equilibrium state generated by reactions back to the attL and attR parental forms under limiting Gp44 concentrations. Stoichiometric requirements of the RDF for the excision reaction by Bxb1 and C31 integrases have also been reported (7,8,37). There is a demonstrable effect by the order of addition of components to LR reactions.…”

Section: Resultsmentioning

confidence: 85%

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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Section: Resultsmentioning

confidence: 85%

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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“…A synapsis inhibition mechanism is not supported by a recent kinetic model described for φC31 Int recombination by Pokhilko et al. (39), where a synaptic LxR complex is predicted to be the most stable species in the P + B → L + R pathway. We note, however, that the model's prediction of distinct reaction intermediates that may differ by the nature of their CC interaction partners is consistent with the structures and models described here.…”

Section: Discussionmentioning

confidence: 99%

Coiled-coil interactions mediate serine integrase directionality

Gupta

Sharp

Yuan

et al. 2017

Nucleic Acids Research

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Serine integrases are bacteriophage enzymes that carry out site-specific integration and excision of their viral genomes. The integration reaction is highly directional; recombination between the phage attachment site attP and the host attachment site attB to form the hybrid sites attL and attR is essentially irreversible. In a recent model, extended coiled-coil (CC) domains in the integrase subunits are proposed to interact in a way that favors the attPxattB reaction but inhibits the attLxattR reaction. Here, we show for the Listeria innocua integrase (LI Int) system that the CC domain promotes self-interaction in isolated Int and when Int is bound to attachment sites. Three independent crystal structures of the CC domain reveal the molecular nature of the CC dimer interface. Alanine substitutions of key residues in the interface support the functional significance of the structural model and indicate that the same interaction is responsible for promoting integration and for inhibiting excision. An updated model of a LI Int•attL complex that incorporates the high resolution CC dimer structure provides insights that help to explain the unusual CC dimer structure and potential sources of stability in Int•attL and Int•attR complexes. Together, the data provide a molecular basis for understanding serine integrase directionality.

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“…We built a mathematical model based on the method described by Colloms et al (10), in which C31 integrase promoted assembly of linear DNA fragments (encoding genes for enzymes of the carotenoid biosynthetic pathway) and a supercoiled plasmid vector (see above). Supercoiled vector was used because of high recombination efficiencies with supercoiled substrates (12). All substrates (fragments and vector) have two att sites (as in Fig.…”

Section: 1model Constructionmentioning

confidence: 99%

“…1S). The model also describes the inactivation of integrase observed in vitro (possibly due to precipitation) (12) and protection of integrase against inactivation upon its binding to DNA (section 3).…”

Section: 1model Constructionmentioning

confidence: 99%

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Mathematical modelling of serine integrase - mediated gene assembly

Pokhilko

Kane

Stark

et al. 2018

Preprint

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Site-specific recombination promoted by serine integrases can be used for ordered assembly of DNA fragments into larger arrays. When a plasmid vector is included in the assembly, the circular product DNA molecules can transform E. coli cells. A convenient "one-pot" method using a single integrase involves recombination between pairs of matched orthogonal attachment sites, allowing assembly of up to six DNA fragments. However, the efficiency of assembly decreases as the number of fragments increases, due to accumulation of incorrect products in which recombination has occurred between mismatched sites. Here we use mathematical modelling to analyse published experimental data for the assembly reactions and suggest potential ways to improve assembly efficiency. We assume that unproductive synaptic complexes between pairs of mismatched sites become predominant as the number and diversity of sites increase. Our modelling predicts that the proportion of correct products can be improved by raising fragment DNA concentrations and lowering plasmid vector concentration. The assembly kinetics is affected by the inactivation of integrase in vitro. The model also predicts that the precision might be improved by redesigning the location of attachment sites on fragments to reduce the formation of the wrong circular products. Our preliminary experimental explorations of assembly with C31 integrase confirmed that assembly efficiency might be improved. However, optimization of efficiency would require more experimental work on the mechanisms of wrong product formation. The use of a more efficient integrase (such as Bxb1) might be a more promising approach to assembly optimization. The model might be easily extended for different integrases or/and different assembly strategies, such as those using multiple integrases or multiple substrate structures.

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The mechanism of ϕC31 integrase directionality: experimental analysis and computational modelling

Cited by 21 publications

References 33 publications

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

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

Coiled-coil interactions mediate serine integrase directionality

Mathematical modelling of serine integrase - mediated gene assembly

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