Switching the polarity of a bacteriophage integration system

Smith, Martha; Till, Rob; Smith, Margaret Caroline MacHin

doi:10.1111/j.1365-2958.2003.03942.x

Cited by 57 publications

(80 citation statements)

References 34 publications

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“…Random collision of two att sites within a supercoiled substrate molecule leads to many different synapse topologies, and thus a complex mixture of product topologies (Fig. 1B) (19,20). To restrict the topology of the ϕC31 system, we made substrates containing a pair of hybrid "phes" recombination sites.…”

Section: Resultsmentioning

confidence: 99%

“…Repeated rounds of reaction and/or persistently cleaved intermediates could be disadvantageous, leading to genetic instability. Previous in vitro analysis of ϕC31 integrase activity is consistent with single-round reactions and efficient ligation of recombinant sites (19). We therefore set out to design a system that would allow us to detect single or repeated rounds of ϕC31 integrase-mediated recombination by topological analysis of the reaction products, and thus determine whether the mechanism is gated (that is, preferentially terminates after a single round of strand exchange) or ungated, involving a rotationally open intermediate.…”

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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: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…Although we have not demonstrated the minimum sequence required for the use of attB Cd by TndX, 50 bp was sufficient to maintain site-specific insertion by Tn5397 in B. subtilis. This is comparable to the attachment sites used by the phage-encoded serine integrases, such as C31, Bxb1, and RV1 (4,11,16,30,31,32). The ability to switch the polarity of insertion of Tn5397 by changing the crossover sequence in attB Cd to 5Ј TC is also strongly reminiscent of the phage integrases and implies that the same mechanism of recombination is employed by TndX.…”

Section: Discussionmentioning

confidence: 72%

The Conjugative Transposon Tn 5397 Has a Strong Preference for Integration into Its Clostridium difficile Target Site

2006

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Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB Cd , in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5 GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB Cd was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB Cd . This experiment was repeated using a 50-bp attB Cd with no loss of target preference. The mutation of the 5 GA to 5 TC in the attB Cd target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB Cd , attL, attR, attTn, and attB Bs3 with relative binding affinities attTn Ϸ attR > attL > attB Cd > attB Bs3 . We conclude that TndX has a strong preference for attB Cd over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.Conjugative transposons are genetic elements that can mobilize via transposition and conjugation from the genome of a donor to that of a recipient, sometimes across large phylogenetic distances. As they commonly encode antibiotic resistances, they are clinically important (7,22,24,27,29). Tn5397 is a 21-kb tetracycline resistance-encoding conjugative transposon originally found in Clostridium difficile (13,20,21). It can transfer from C. difficile to Bacillus subtilis and back to C. difficile (20). It can also transfer in a model oral biofilm community, indicating that the element is likely able to transfer in natural environments (26).Tn5397 has been completely sequenced, revealing that it is very closely related to the extensively studied, conjugative transposon Tn916 in the regions concerned with transfer and resistance to tetracycline (25). However, the regions required for transposition in Tn916 and Tn5397 are completely different. The insertion and excision of Tn5397 are dependent on the large serine recombinase TndX, the only Tn5397-encoded protein required for these functions (33, 34). Tn916, on the other hand, requires the tyrosine recombinase (Int) for integration and Int and the accessory factor Xis for excision (15). Although Tn916 can insert into multiple sites in most hosts, it does have preferred integration sites and, in some strains of C. difficile, it has one highly preferred site (33). The clostridia also c...

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“…In Synechococcus spp., the transcription of the phageencoded genes ensures unabated photosynthetic capacity in the phage-infected cell. M. Smith (Aberdeen, United Kingdom) reported on a novel serine recombinase from the Streptomyces phage phiC31 (86,87). Although DNA excision is normally believed to require helper proteins, gain-of-function mutants were isolated that can perform both integration and excision in their absence.…”

Section: Genomics Evolution and Bacteriophagementioning

confidence: 99%