388 Biochemical Society Transactions (2010) Volume 38, part 2

Site-specific recombination by φC31 integrase andother large serine recombinasesMargaret C.M. Smith*1, William R.A. Brown†, Andrew R. McEwan* and Paul A. Rowley**Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, U.K. and †Institute of Genetics, University of Nottingham, Nottingham

NG7 2UH, U.K.

AbstractMost temperate phages encode an integrase for integration and excision of the prophage. Integrases belongeither to the λ Int family of tyrosine recombinases or to a subgroup of the serine recombinases, the largeserine recombinases. Integration by purified serine integrases occurs efficiently in vitro in the presenceof their cognate (∼50 bp) phage and host attachment sites, attP and attB respectively. Serine integrasesrequire an accessory protein, Xis, to promote excision, a reaction in which the products of the integrationreaction, attL and attR, recombine to regenerate attP and attB. Unlike other directional recombinases,serine integrases are not controlled by proteins occupying accessory DNA-binding sites. Instead, it is thoughtthat different integrase conformations, induced by binding to the DNA substrates, control protein–proteininteractions, which in turn determine whether recombination proceeds. The present review brings togetherthe evidence for this model derived from the studies on φC31 integrase, Bxb1 integrase and other relatedproteins.

IntroductionAfter infection of a sensitive host cell, temperate bacterio-phages ‘make a decision’ between lytic growth and lysogeny,a latent state in which phage lytic genes are repressed and thephage genome is often integrated into the host chromosome[1]. Lysogeny is reversible, and a minority of cells withina population of lysogens will spontaneously lyse, releasingprogeny phage. Allan Campbell [2] first proposed that the λ

prophage is integrated into the host chromosome at a specificsite by recombination, a process that requires specialized‘attachment sites’, attP and attB, in the phage and hostchromosomes respectively [3]. The integrated prophage isflanked by attL and attR, which are the substrates for excisiverecombination, a process that reforms attP and attB andreleases the prophage.

Phage integrases within the serinerecombinase familyUntil the mid-1990s, all known phage integrases were relatedto the Escherichia coli K12 phage λ integrase. The λ Int pro-teins belong to the large and diverse family that use a tyrosineresidue to generate a transient 3′ phosphotyrosine link to storethe energy of the phosphodiester bond during recombination[3,4]. In contrast, the integrases from the Streptomyces phagesφC31 and R4, the lactococcal phage TP901-1 and SpoIVCA,a recombinase that excises a prophage-like element from the

Key words: bacteriophage, excision, φC31 integrase, integration, protein–DNA interaction,

protein–protein interaction, serine recombinase.

Abbreviations used: CTD, C-terminal domain; NTD, N-terminal domain; RDF, recombination

directionality factor.1To whom correspondence should be addressed (email maggie.smith@abdn.ac.uk).

Bacillus genome during sporulation, were found to be relatedto the resolvase/invertases and therefore belong to the serinerecombinase family of site-specific recombinases [5–8]. Theserine recombinases are evolutionarily and mechanisticallydistinct from the λ Int family as they use a serine residueas a nucleophile during catalysis [4]. Recombination by theresolvase/invertases occurs within a tetramer of recombinasesubunits bound to two DNA substrates [4]. A concerteddouble-strand break in both substrates occurs with theconcomitant formation of covalent 5′ phosphoserine links tothe recombinase subunits and an overhanging 3′ dinucleotide.Strand exchange is thought to involve a mechanism in whichtwo recombinase subunits covalently bound to half-sites fromdifferent DNA substrates are exchanged by a 180◦ rotationwith respect to the other two subunits [9–11]. This rotationmechanism brings together the cleaved ends of the two half-sites that will comprise the products; the ends are joined andintegrase is released. In the resolvase/invertases, the catalysisand strand exchange is driven by the NTD (N-terminaldomain).

The sequence similarity between the phage-encoded serineintegrases with the resolvase/invertases extends only so faras the catalytic NTDs, and it is clear that these proteinsuse the same mechanisms of DNA cleavage and DNA strandexchange as the resolvase/invertases [7,12–17]. In the placeof the short ∼40-amino-acid CTD (C-terminal domain) inthe resolvase/invertases, SpoIVCA and the phage-derivedserine integrases have much larger C-terminal domains (∼300to >500 amino acids) and so earned a collective term, thelarge serine recombinases [18]. Alignments of the large serinerecombinases show that, although they are a diverse groupof proteins, there are common motifs in the CTDs, mostly

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Machines on Genes: Enzymes That Make, Break and Move DNA and RNA 389

Figure 1 Conserved motifs in φC31 and Bxb1 integrases and in the transposase, TnpX

The horizontal black line is a linear representation of the amino acid sequence. Five conserved motifs identified either by

sequence alignments or a structural prediction algorithm, COILS [34,59], are colour-coded: green, pfam0239, the resolvase

NTD; purple, pfam07508; yellow, the putative zinc finger; blue, putative coiled-coil structural motif; orange, a conserved

isoleucine-, leucine- and valine-rich region). The sequence co-ordinates for each motif in the three proteins are indicated.

No coiled-coil motif is included on Bxb1 integrase because, although the COILS program found a candidate motif, it fell

short of the statistical criteria. The white boxes summarize structure–function experiments in which protein fragments

have been prepared and their in vitro activities reported [15,39,44,60]. For each protein, specific amino acid substitutions

are also indicated which have revealed structure–function information. Abbreviations: N, N-terminus; C, C-terminus; PSS,

protease-sensitive site; PI, protein interaction activity.

of unknown function (Figure 1) [18]. The CTD is, however,known to be required for DNA binding and for the controlof integration against excision (see below).

The incidence of the large serine recombinases appears tobe enriched in phages that infect actinobacteria and in the

mobile genetic elements of the Gram-positive bacteria. Theconjugative transposon Tn5397 from Clostridium difficileand the mobilizable transposon Tn4451 in Clostridiumperfringens both contain large serine recombinases, TndXand TnpX for integration and excision respectively of

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390 Biochemical Society Transactions (2010) Volume 38, part 2

the elements [19–21]. The SCC elements (staphylococcalchromosomal cassettes) that encode the clinically importantmethicillin-resistance gene, mec, are also integrated andexcised via large serine recombinases [22].

The default activity of phage-encodedserine integrases is integrationSeveral of the phage-encoded serine integrases have beenexpressed in E. coli and purified for use in in vitrorecombination assays and/or DNA-binding assays [13,23–28]. The integration reactions mediated by φC31, Bxb1,φRv1, φBT1 and φFC1 integrases require a simple buffer,the substrates (attP and attB), which can be on the same orseparate, supercoiled or linear molecules, and no additionalenergy sources. The recombination products attL and attRcomprise a half-site from attP and a half-site from attB ina reciprocal format. The minimal recombination sites aregenerally less than 50 bp for both attP and attB [14,23,25–27,29,30]. This contrasts with the phage λ integration systemin which a 240 bp attP site contains the core and arm-bindingsites for λ Int and the accessory protein-binding sites [3]. Thereaction conditions for the serine integrases are also differentfrom those of the resolvase/invertases that require supercoiledDNA with both recombination sites located on thesame DNA molecule [4,31,32].

At first glance, the properties of the phage serine integrasesin these in vitro recombination reactions resemble those forthe bidirectional recombinase Cre [33]. However, all of theserine integrases are unidirectional in the absence of otherproteins [13,24,25,27]. Indeed, even using a highly sensitivein vitro recombination assay for φC31 integrase activity,no recombination could be detected between any pair ofrecombination sites other than attP and attB [34]. In fact,serine integrases require an accessory protein, an Xis orRDF (recombination directionality factor), to mediate theexcision reaction between attL and attR to produce attPand attB [23,35,36]. At the mechanistic level, how doesintegrase discriminate between the four attachment siteswithout resorting to the use of accessory binding sites asseen with other directional recombinases?

Distinct Int–attP and Int–attB interactionsenable synapsis during integrationFor integration to occur, integrase must recognize and bindto attP and attB and bring these sites together in a synapsebefore DNA cleavage, strand exchange and ligation of theproducts (Figure 2). φC31 and Bxb1 integrases are dimers insolution and bind to the attachment sites with one subunitcontacting each half-site [30]. The bound dimers then bringattP and attB together in a synaptic complex that probablycontains a tetramer of integrase subunits [15] (Figure 2).With φC31 integrase (in this case, a catalytic mutant ofintegrase IntS12A), the synaptic complexes are observedwhen integrase is provided with attP and attB [16]. Withnative φC31 integrase and attP/attB as substrates, there is

Figure 2 Model for integration by φC31 integrase

Integrase (Int) is shown as a two-domain protein with the dark-coloured

rounded rectangle as the large CTD and the semi-transparent

pale-coloured cut-off ellipse as the NTD. (a) Int in free solution is a

dimer. (b) When Int binds to attP and attB (indicated by black and grey

lines respectively), it adopts attP- or attB-bound conformations shown

by the different colours (blue and red respectively) and by the activation

of a protein–protein interaction motif (probably the coiled-coil motif; see

the text) in the CTD. (c) The protein–protein interaction domain in the

attP bound Int interacts with the CTD of the attB-bound Int to form a

tetramer. (d) Formation of the tetramers via the CTD interaction could

then cause the NTDs to interact, stimulating a conformational change in

the NTDs (reminiscent of that in γ δ resolvase on synapsis [10]), which

activates (e) DNA cleavage and (f) strand exchange. (g) Joining of the

phosphodiester backbone in the products attL and attR causes Int to

adopt an asymmetric dimer conformation which disables synapsis and

prevents excision in the absence of an accessory protein, Xis. Adapted

from [39] with permission. c© 2009 Oxford University Press.

little accumulation of synaptic complex, and it is likely thatactivation of DNA cleavage and strand exchange occurs veryrapidly compared with the rate of formation of the synapse[34,37].

The critical stage of recombination where sites are selectedfor recombination is at synapsis. φC31 IntS12A, in theabsence of any other proteins, cannot synapse any pair ofattachment sites other than attP and attB, including, forexample, two attP or attB sites, or attR with attL. Theselection of sites for synapsis is not due to differences ofaffinity [15,30]. The favoured hypothesis is that integraseadopts distinct conformations when bound to attP and attBthat enable the formation of synaptic tetramers (Figure 2)[15,16,30]. Mutations in φC31 attB can severely reduce therate of synapsis without affecting binding affinity, and this isconsistent with a mechanism in which DNA contacts inducespecific conformational changes [38].

DNA recognition in the large serine recombinases occursthrough the CTDs. The CTDs from Bxb1 and φC31 have

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Machines on Genes: Enzymes That Make, Break and Move DNA and RNA 391

been purified as isolated domains and shown to bindspecifically to their cognate attP and attB sites [15,39]. Inboth cases, the isolated CTDs are monomers in free solution,but the φC31 CTD binds co-operatively to attB (and attLand attR), but not to attP, indicating that integrase recognizesattB and attP differently [39]. This is perhaps not surprisinggiven that the sequences of attP and attB are very differentfrom each other (see, for example, [18]).

Structure–function studies have not revealed a commonDNA-binding motif within the large CTDs (Figure 1).A fragment of Bxb1 integrase containing the NTD and asubdomain of the CTD having homology with the conserved‘recombinase’ motif (pfam07508) binds with low affinity[15]. A mutation in a linker that joins the NTD and CTDin Bxb1 integrase reduces binding to attB, but not toattP, and this may be the first clue that different motifsof integrase interact with different attachment sites [15].Moreover, the phenotype of this mutant adds further to theevidence for different integrase conformations when boundto attB and attP. Structure–function analysis with TnpXshowed that this protein has two regions at the extreme C-terminus involved in DNA binding. The most C-terminalof these two DNA-binding motifs can be removed, andthe protein still functions as a transposase, suggesting thatthis motif has an accessory function. The second DNA-binding motif is not in a conserved region, and so it isnot clear whether other recombinases have an equivalentfunction at this position. Although mutations in TnpX thatmap in the region with homology with pfam07508 can affectbinding, a fragment containing this region does not bind toDNA [40].

This discussion highlights how little we understand abouthow the serine integrases recognize their sites, both in termsof the protein motifs that are used to contact DNA and thesequence requirements in the attachment sites themselves.

Protein–protein interactions betweenintegrase subunits control synapsisThe serine integrases bring attP and attB together to forma putative synaptic tetramer during the integration reaction.In solution, φC31, Bxb1 integrases and the isolated NTDsfrom Bxb1 and TP901-1 integrases are dimers [15,30,39,41].The structure of the TP901-1 NTD shows that the serineintegrases dimerize via a long α-helix (the E-helix) that, in theresolvase/invertases, also mediates dimer formation [41,42].

The NTDs also mediate the interactions that bring therecombinase dimers together to form a synaptic tetramer atthe crossover site [4,10,41]. Structural studies of resolvaseand the NTD of TP901-1 integrase provide details of thesynaptic interface at the atomic level [10,41]. The formationof the synaptic interface through the NTD–NTD interactionsactivates DNA cleavage and is therefore a critical stagein all serine recombinase reactions [4]. In φC31 integrase,Val129 is equivalent to residues in resolvase, Hin invertaseand in TP901-1 NTD that have been shown to contributeto the NTD synaptic interface [10,37,41,43]. Substitutions

in φC31 integrase can affect synapsis (V129A) and DNAcleavage (V129G), phenotypes that strongly suggest thatthis interface is both conserved and critical for integraseactivity.

Recent data show that the CTD also has a role in thesynapsis of substrates in φC31 integrase [39]. The isolatedφC31 integrase CTD, a monomer in free solution, binds attPand attB and brings them together to form a putative synaptictetramer. The CTD synapse echoes the synapse made byfull-length integrase, as both complexes only form with attPand attB. Studies on hyperactive mutants of φC31 integraseprovide some insights on the role of the CTD synapticcomplex [34,39]. Mutants of φC31 integrase were isolatedthat could recombine attL×attR [34]. IntE449K is similarto native integrase in attP×attB recombination activity, butcan also synapse and recombine attL×attR (and reactions thatmimic excision, i.e. attL×attL and attR×attR). It seems likelythat E449K disrupts a mechanism that normally preventsattL×attR synapsis. The E449K substitution in the contextof the isolated CTD prevented the formation of the CTDsynapse. This suggests that the CTD synapse may be requiredto remove a block on the formation of the NTD synapticinterface. Thus IntE449K is free to synapse attL×attR,attL×attL, attR×attR in addition to attP×attB entirely viaNTD interactions. The observation that E449K partiallyrescues a mutation in the NTD (V129A) that reduces therate of formation of the synaptic complex is consistent withthe idea that the NTD and CTD interact to control synapsis[37].

A coiled-coil motif in the large CTDmediates protein–protein interactionsAll of the mutants that showed any hyperactivity wereclustered in the region 449–485 (Figure 1), where structurepredictions indicate that φC31 integrase (and several otherserine integrases) may adopt a coiled coil [34]. These motifsare often used for protein–protein interaction and a constructcontaining this region fused to MBP (maltose-bindingprotein) was able to oligomerize [39]. A fragment of TnpXcontaining the equivalent region could also oligomerize [44](Figure 1). In φC31 integrase, the coiled-coil motif appearsto have a role in formation of the CTD synaptic complex,as substitutions that disrupt the protein–protein interactioncannot form the CTD synaptic tetramer. However, thesesubstitutions also disrupt DNA binding and/or co-operativebinding. The current model proposes that the coiled-coilmotifs mediate an interaction between CTDs bound toadjacent half-sites in attB, attL and attR, whereas the coiled-coil motifs in the CTDs bound to attP are poised to nucleateinteractions between subunits bound to attP and attB, leadingto the formation of the CTD synaptic tetramer. This tetramer,which could be transient in the presence of the NTDs, thenreleases the inhibition of the synapsis via the NTDs. Thismodel explains the co-operative/non-co-operative DNAbinding by the isolated CTD and the phenotypes of aminoacid substitutions leading to hyperactivity.

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Integrase complexes with attP and attBare functionally symmetrical andasymmetrical with attL and attRThe hyperactive φC31 integrase mutant IntE449K permitteda comparison of the integration and excision reactions [17,34].As attP and attB contain imperfect inverted repeats centredaround the 2 bp crossover site, they contain two P-type andtwo B-type arms respectively, whereas attL and attR containone B-type and one P-type arm in a reciprocal arrangement.By manipulating the sequence at the 2 bp crossover site, thenature of the synaptic complex can be inferred by studyingthe products of recombination [14,17]. To date, the 2 bpcrossover sequences in the substrates for every large serinerecombinase so far studied are non-palindromic. This is fora good reason, as it ensures that, during integration, the leftarm of attB becomes joined to the right arm of attP andvice versa to form attL and attR respectively [14,17]. If the2 bp crossover sequence is made to be palindromic, aberrantproducts are observed where the joining is left arm to leftarm and right arm to right arm and this occurs at the samefrequency as formation of the normal products. The twotypes of products are observed because two types of synapseare constructed that differ by the parallel or antiparallelalignment of the substrates. This can only occur if integrasedimers bound to attP and attB are functionally symmetrical.Using the same approach for the attL×attR synapsis withthe hyperactive φC31 integrase mutant, a preferred excisionsynapse is inferred in which integrase subunits bound to P-type arms contact integrase subunits bound to B-type armsat the tetramer interface [34]. This implies that the integrasedimers bound to attL and attR are functionally asymmetrical.

The formation of the synaptic tetramer is thereforeinfluenced by the half-sites to which integrase subunits arebound. It is worth emphasizing, however, that the integrasesubunits do not act independently of each other in adopting aconformation that will enable a subunit–subunit interaction,as even with the hyperactive mutant, the selection of DNAsubstrates to synapse (and recombine) is not indiscriminate.Synapsis is only observed where the substrates collectivelycontain two B-type and two P-type arms. This argues that theconformation of the integrase dimer bound to the attachmentsite is the unit that determines whether a synaptic interface ismade.

The role of Xis in excisionThe serine integrases, like those that belong to the tyrosineintegrase group, recruit an Xis or RDF to enable excisionof the prophage [23,35,36,45]. Xis has been identified forthree phages, TP901-1, φRv1 and Bxb1, and there is nodetectable sequence conservation between them. TP901-1 Xis(encoded by orf7 on the TP901-1 genome) is a 64-amino-acidbasic protein whose only homologue in the database is fromanother lactococcal phage which also has a serine integrase[35]. φRv1 gpXis is also small (73 amino acids) and basic,and many mycobacteriophage genomes encode homologues[23]. The Bxb1 RDF, gp47, is 255 amino acids in length and

has a conserved domain in the protein phosphatase family[36]. Close homologues of Bxb1 gp47 are present in othermycobacteriophages, including those that carry a tyrosineintegrase (e.g. L5), and it has been suggested that this proteinmay have a function in addition to its role as an RDF. In theλ Int family, the genes encoding integrase (int) and Xis (xis)are normally neighbours. In φRv1, TP901-1 and Bxb1, thegenes encoding integrase and Xis are separated by one, elevenand five genes respectively [23,35,36]. The diversity of the Xisproteins and the random location of the Xis-encoding genessuggest that phage proteins could be recruited to controlintegrase directionality on a rather ad hoc basis.

Studies on the biochemical mechanism of the RDFfrom Bxb1 indicate that this protein controls integrasedirectionality by protein–protein interactions. Unlike otherRDFs, Bxb1 gp47 does not bind to DNA, and the minimalsites for attL×attR recombination are ∼50 bp [36]. Bxb1gp47 recognizes and binds tightly to integrase bound toattP and attB, which may explain the inhibition by gp47 onintegration. Although gp47 binds less strongly to integrasebound to attL and attR, it stimulates formation of theintegrase/attL/attR synapse. Hatfull and colleagues have alsodemonstrated the same structural bias in the formation ofexcisive synaptic complexes as seen with the hyperactiveφC31 integrase described above [45]. The bias in the align-ment of the substrates at synapsis combined with thenon-palindromic nature of the 2 bp at the crossover siteprevents unwanted attL×attL or attR×attR recombinationand ensures the correct use of attL×attR for prophageexcision [45].

Behaviour of φC31 integrase in theeukaryotic cellThe in vitro properties of φC31 integrase prompted itsapplication as a tool for genome engineering in heterologoussystems, and its activity has been demonstrated in manyeukaryotic organisms [29,46–50]. The irreversible activity ofφC31 integrase has enabled efficient integration and inversionof DNA segments, manipulations which are either inefficientor incomplete with a bidirectional recombinase such as Cre[51,52]. Precise genome engineering requires the insertionof the cognate attP and attB sites. However, it appears thatattachment site recognition by integrase is sufficiently relaxed[53] to permit the use of endogenous sequences in the humanand mouse genome as integration sites in a novel approachto gene therapy [54,55]. This ‘ectopic’ recombination occursat very low frequency and integrase targets many so-called‘pseudo-attP’ sites within the human and mouse genomes[29,52,56]. Despite these problems, it is has been shown thatφC31 integrase may be ‘improved’ by protein engineering torecognize the pseudo-sites more efficiently [57].

The behaviour of φC31 integrase in eukaryotic cellshas contributed to our mechanistic understanding of theseproteins. Integrase-mediated attP×attB recombination inchick or in human cells lines often leads to small deletionsin the recombination sites [52]. It was proposed that these

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Machines on Genes: Enzymes That Make, Break and Move DNA and RNA 393

deletions were the result of NHEJ (non-homologous end-joining) enzymes detecting the recombination intermediatecontaining integrase covalently bound to the cleavedsubstrates, which are then repaired. If this is the case, thenit implies that the rotation mechanism that drives strandexchange may be much slower in the nuclear environment,possibly due to the presence of nucleosomes and/or differentDNA topology compared with that in the prokaryoticnucleoid. With naked supercoiled substrates, φC31 integrasedoes not cause accumulation of cleaved intermediates, butthey are easily detected when both substrates are linear.This suggests that negative supercoiling does stimulate eitherstrand exchange or joining of the products [34].

A second observation is the preference for one substrate(attP) over the other (attB) as the docking sites inheterologous systems [29]. The bias for this is not clear,but one possibility is that there are cellular proteins thatinteract with and inhibit integrase bound to attB, but notto attP, possibly due to different conformations of integrasebound to the two sites. In support of this a human protein,DAXX (death-domain-associated protein), has been shownto interact with the coiled-coil motif, which as describedabove has profound effects on integrase activity [34,58].

Funding

Funding for research in the M.C.M.S. and W.R.A.B. laboratories is from

the Biotechnology and Biological Research Council [grant numbers

BB/D007836/1 and BB/E000894/1].

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Received 24 July 2009doi:10.1042/BST0380388

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