Synapsis in Phage Bxb1 Integration: Selection Mechanism for the Correct Pair of Recombination Sites

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Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase–DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase–DNA complexes.

Introduction

Mycobacteriophage Bxb1 is a temperate phage that forms stable lysogens in Mycobacterium smegmatis.1 Its genome sequence reveals that it shares a similar genomic architecture with mycobacteriophages L5 and D29, and these phages have many genes in common.1, 2, 3 Prophage formation by Bxb1 is accompanied by integration of the Bxb1 genome into the M. smegmatis chromosome, which is catalyzed by integrase-mediated, site-specific recombination between phage (attP) and bacterial (attB) attachment sites. Whereas both phages L5 and D29 utilize lambda-like tyrosine-integrases, Bxb1 uses a serine-integrase that belongs to the large family of serine-recombinases that includes transposon resolvases (e.g. Tn3 and γδ resolvases) and DNA invertases (e.g. Hin, Gin etc.).4, 5

The serine-integrases represent a particularly interesting subset of the serine-recombinase family. More than 20 of these have been identified,6 and in vitro systems have been established for Bxb1,7 Streptomyces phage ϕC318 and the Mycobacterium tuberculosis prophage-like element, ϕRv1.9 All of the serine-integrases appear to be composed of an ∼150 amino acid residue N-terminal segment that is related to the catalytic domain of transposon resolvases, and a large C-terminal region 300–600 residues in length. The specific functions of these C-terminal segments are not known, although they are highly varied in their amino acid sequences and are likely to be important for DNA binding. The similarity of the N-terminal segments to transposon resolvases suggests that they share a common catalytic mechanism, and in the Bxb1 and ϕC31 systems it has been shown that a serine residue close to the N terminus (S10 and S12, respectively) is required for recombination;7, 8, 10 strand exchange involves a concerted double-strand (ds) break about the central dinucleotide, and covalent linkage between integrase and DNA.7, 11

The serine-integrases differ from resolvases and DNA invertases in that the recombination substrates (attP and attB) are different from each other in both sequence and length. Phage Bxb1 integrates into an attachment site (attB) located within the M. smegmatis groEL1 gene,12 and the sequences required for attB function are located within a 38 bp DNA segment of the chromosome; attP functions lie within 48 bp of the Bxb1 genome (Figure 1(a)). The requirement for relatively small DNA sites is seen also in the ϕC3113 and ϕRv1 systems,9 and in all three systems, not only are there distinct size requirements for attP and attB, but these sites are considerably different at the sequence level (Figure 1(a)). This raises the question of how serine-integrases recognize the different attP and attB sites, and how these are distinguished from the product sites, attL and attR, each of which consists of one half-site from each of attP and attB (Figure 1(a)). Furthermore, all three systems are strongly directional and integrase alone does not promote excisive recombination using attL and attR as substrates. In the ϕRv1 system, a prophage-encoded protein has been identified that is required for excision,14 but similar activities have yet to be described for either ϕC31 or Bxb1. However, it has been reported that the failure of the ϕC31 integrase to perform excision results from the inability of the integrase to promote synapsis between attL and attR DNAs.10, 11

The simplicity of the DNA substrates makes the serine-integrase reactions distinct from the DNA-invertase and resolvase systems, which require either additional sites and factors, or multiple recombinase binding sites. Moreover, these systems typically employ DNA topology as a mechanism for determining whether the DNA substrates are correctly configured to undergo strand exchange.4, 5 In the Bxb1 integration system there is no requirement for DNA supercoiling, complex sites, or protein cofactors, although there is a strict requirement for the correct pair of sites (i.e. attP and attB) to initiate the process of strand exchange.7, 12 However, synapsis between attP and attB occurs with the sites in either parallel or antiparallel alignments, and it is only the asymmetric nature of the central dinucleotide (5′-GT) that dictates the polarity of integration;7 a similar situation occurs in the ϕC31 system.15 Thus, with respect to synapsis, attP and attB are functionally symmetrical, even though they exhibit only partial symmetry at the sequence level (Figure 1(a)). This poses a conundrum for excisive recombination in these systems, since attL and attR (each of which consists of a B-type and P-type half-site; Figure 1(a)) must be distinguished from each other in order to enable attL×attR recombination and exclude attL×attL or attR×attR recombination.7

A subunit exchange model for strand exchange has been proposed for the Tn3/γδ resolvase and the Hin invertase systems.16, 17, 18 In this model, a recombinase tetramer initiates strand cleavage and covalent linkage of the protein to the DNA. During strand exchange, the protein–protein interactions joining the dimers at the recombination sites must be broken, and the two protein-bound half-sites rotate 180°, followed by ligation of the half-sites and formation of new protein dimers. The overall reactions are highly concerted and cleaved half-sites in their free form are not observed in the wild-type systems. It seems likely that the serine-integrases utilize a similar mechanism for strand exchange, and while there are no accessory sites or proteins to hold the complex together during strand exchange, it is plausible that the large C-terminal domain provides this function. Nevertheless, these proteins are expected to have unusual DNA-binding properties, in that they must accommodate four different sites with different sequences, sizes and geometries, and the conformations of the different DNA complexes are likely to play critical roles in determining which pairs of sites are permissible for recombination. Bxb1 gpInt must therefore have the capacity to discriminate both structurally and functionally between different attachment site identities.

Here, we show that Bxb1 gpInt binds to all four DNA substrates (attP, attB, attL and attR) but promotes synapsis of only attP and attB, each bound by a dimer of gpInt. Thus, the selection of recombinationally permissive pairs of protein–DNA complexes is a two-step process, requiring identification of attachment site DNA through sequence recognition, followed by the discrimination between different protein–DNA complexes. This second step likely arises from conformational changes induced upon DNA binding, such that attP-bound and attB-bound dimers are the only pair that can undergo synapsis and subsequent strand exchange. This is supported by the observations that mutant substrates are competent for DNA-binding but not recombination, and that a substantial portion of the binding energy is invested in conformational changes of the protein–DNA complexes. Furthermore, these proposed conformational changes require covalent linkage of the two half-sites within attP and attB, even though the binding of the C-terminal DNA-binding domain does not. A mutant protein that is specifically defective in attB binding and in synaptic complex formation illustrates the need for gpInt to adopt different conformations at different att-sites.

Section snippets

Bxb1 gpInt binds attB, attP, attL and attR but synapses only attB with attP

Bxb1 gpInt catalyzes integrative recombination efficiently between attP and attB, and the integration reaction is strongly directional, such that no recombination occurs between attL and attR.7 We have tested all ten possible combinations of the four sites, and only attP×attB recombination was observed (data not shown). There are two plausible explanations for why pairs of sites other than attP×attB fail to recombine. First, gpInt could bind with distinctly different affinities to each site,

Discussion

Serine-integrases represent a particularly interesting sub-family of the serine-recombinases. All of the serine-recombinases must overcome two problems in substrate selection. First, they must discriminate specific sites from non-specific DNA, which for the transposon resolvases and DNA-invertases, requires recognition of a single DNA sequence that, when repeated in inverted orientation, forms the substrate sites. Second, synapsis must be regulated such that only the correct pair of sites is

DNA substrates and oligonucleotides

DNA fragments (200 bp) containing attP, attB, attL and attR were amplified by PCR and cloned into the EcoRV site of pMOS Blue (Amersham) to obtain pMOS-attP, pMOS-attB, pMOS-attL and pMOS-attR. Fragments of 106 bp containing each of the above sites were amplified by PCR and 5′-end labeled using phage T4 polynucleotide kinase (PNK) for use in the gel-retardation assays.

DNA fragments (50 bp) containing wild-type attP and attB were prepared by annealing complementary oligonucleotides. Mutant attB

Acknowledgements

We thank Molly Scanlon for excellent technical support, Lori Bibb for helpful comments on the manuscript, and John Hempel for the N-terminal sequence analysis. This work was supported by NIH grant AI59114.

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