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Journal of Bacteriology, May 2006, p. 3409-3411, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3409-3411.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of Secondary Attachment Sites in Changing the Specificity of Site-Specific Recombination

Edit Rutkai,¹ Andrea György,¹ László Dorgai,^1* and Robert A. Weisberg²

Department of Molecular Biotechnology, Bay Zoltán Institute for Biotechnology, Szeged, Hungary,¹ Section on Microbial Genetics, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland²

Received 24 October 2005/ Accepted 10 February 2006

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We previously proposed that lambdoid phages change their insertion specificity by adapting their integrases to sequences found in secondary attachment sites. To test this model, we quantified recombination between partners that carried sequences from secondary attachment sites catalyzed by wild-type and by mutant integrases with altered specificities. The results are consistent with the model, and indicate differential core site usage in excision and integration.

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Many temperate bacteriophages integrate into the host genome by site-specific recombination. Typically, different phages use different attachment sites, and these sites are frequently unique in both the phage and host genomes (attP and attB, respectively). In many cases, recombination is catalyzed by a phage-coded enzyme, integrase (Int), that belongs to the tyrosine recombinase family (1). The structural similarities of different Ints argue that different site-specificities evolved from a common ancestor.

To explain how a new specificity evolves from an existing one, we proposed a chromosome jumping model (5) (Fig. 1). Key features of this model include phage insertion at a secondary host attachment site (attB*), followed by abnormal prophage excision to produce a transducing phage with a prophage attachment site (attR*) and a complete int gene. attB* eventually becomes the new primary bacterial attachment site, and attR* becomes the new phage attachment site. During this transition, mutations adapt Int to the new sites and vice versa. Secondary sites contain sequences that can be identified with the two core Int binding sites of attB and the 7-bp "overlap region" that separates them. Although the overlap region is not directly recognized by Int, it nevertheless plays an important role in recombination because sites with different overlap regions recombine poorly (6). Individual secondary sites are poor recombination substrates because of sequence differences in the Int binding sites and/or overlap regions.

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FIG. 1. The chromosome jumping model (5). Phage insertion at a secondary site (attB*) is followed by the formation of an attR* transducing phage. Mutations subsequently convert attR* to attP*, a new phage attachment site, and adapt Int (int^) to attB* and attP* (see text). Bacterial DNA is represented by open boxes except for attB*, which is indicated by a striped box. Phage DNA is represented by stippled boxes except for attP, which indicated by a black box. The protein binding sites in attR* (enlarged) are labeled as follows: P and P', arm-type Ints; H and H', integration host factor sites; X, Xis; F, Fis; C and B*', core-type Ints. O is the overlap region. The size of the inserted bacterial DNA in the transducing phage is variable, as is that of the retained DNA (bracketed) from the P' arm of attP.

In this work we examined three features of the model using att sites and int mutants of phage . First, attB* is predicted to become the new integration target, assisted by the overlap identity between attB* and attR*. To test part of this prediction, we measured the effect of overlap region identity in integrative and excisive recombination involving attP and attB* sites. Although previous work strongly implies that overlap region identity promotes recombination involving secondary sites, the effect has not been quantified (5). Second, Int will adapt to the new target by accumulating mutations that increase both recombination frequency and specificity. To test this prediction, we used Int mutants with altered specificities. These mutants increase recombination between att sites of the -related phage HK022 and change secondary site utilization by attP (3, 5). Third, the attR* transducing phage will retain the ability to reinsert by Int-promoted recombination at the attB* site from which it came. In such a phage, a host chromosomal substitution separates the core-type Int binding sites and the overlap region from any remaining arm-type Int binding sites (Fig. 1). To determine the effect of such a substitution, we measured the efficiency of integration of a attR transducing phage in which the P' arm-type Int binding sites are displaced by about 3.1 kb from the core.

Table 1 reports recombination frequencies that test the first two predictions described above. We used two attB* sites identified earlier, yahM and sraF, because they were not efficiently used by wild-type (5). The yahM and sraF sites with overlaps that differed from that of the infecting attP phage were inefficient substrates, both in excision and integration, regardless of which site had the attB* overlap and regardless of which integrase was used. The recombination frequencies were below the detection limit (0.1 to 0.5%). The single exception was the wild-type attP x yahM attB* reaction promoted by the quintuple mutant Int. This correlates well with our previous result: this mutant phage integrated at yahM in 38% of the population of secondary site lysogens (5). Overlap identity elevated the level of recombination of the sraF substrates, catalyzed by wild-type Int, to a low but detectable level. However, the values (1% or less) indicate that sraF discriminates against Int, and its core sites are not effectively recognized by it even after the barrier of the unmatched overlaps is removed. This is also true for yahM: only one of the excisive but not the integrative substrates was detectably recombined. Next, we tested if the substrates were more acceptable for Ints with altered specificities. Two Int mutations, E319R and N99D, increase recombination of phage HK022 attachment sites (3). We found that E319R alone and especially together with N99D substantially increased integrative and excisive recombination involving sraF and yahM sites with matching overlap regions. N99D by itself had little or no effect. The triple mutation S282P-G283K-R287K is unable to promote integrative or excisive recombination of sites but, when combined with N99D and E319R, recombines HK022 sites and promotes insertion into secondary sites (3). The quintuple mutant also promoted substantial levels of integrative and excisive recombination involving yahM and sraF sites with matching overlap regions.

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TABLE 1. Recombination of plasmid substrates containing secondary attB core recognition sites

These results are consistent with the chromosome jumping model. Overlap identity, as expected, helps recombination but in a site-dependent manner. It does not necessarily confer an increase high enough to prevent selective disadvantage of the transducing phage as a consequence of its impaired integration ability. One single mutation—which might even be present in Int before the first integration at the secondary site—led to a much more substantial increase in recombination of secondary att sites at a level that is likely high enough for a temperate phage to escape counterselection, allowing time for Int to acquire additional mutations that improve recognition of the new sites and discriminate against the original attB. The Int mutations used in this work were identified in the /HK022 system and together resulted in a change of specificity. We do not suggest that these particular mutations are necessarily part of a natural specificity change, but the results indicate that such change could occur in this way. Excisive substrates tested in this work had either or secondary overlap sequences. The yahM substrate with secondary overlaps was recombined at a significantly lower level by several Ints than its counterpart with overlaps. It is generally believed that for efficient recombination, matching overlaps and not the absolute sequence are required. Azaro and Landy, however, demonstrated in vitro that the purine/pyrimidine composition of the overlap at several positions alters the direction of resolution of the Holliday structure formed by the first-strand exchange (2). The last four nucleotides in the yahM overlap are CCGA instead of the ATAC. Though these authors did not test this specific sequence, it is conceivable that the change in composition leads to the effect observed.

In Table 2 we present results that support the prediction that Int promotes insertion of attR*. The attR site of the phage we used ( bio936) contains a 3.1-kb substitution of host DNA that includes the B' core Int binding site (Fig. 1). The host DNA replaces a 37-bp segment of attP extending rightwards from the overlap region, thereby deleting the H' integration host factor binding site and moving the P' arm-type Int binding sites about 3.1 kb farther from the core than they are in attP (R. A. Weisberg and E. Rutkai, unpublished results). This phage can be viewed as an attR* transducing phage in which Int and the core sites are already adapted to each other. Int promoted a low but easily detectable frequency of insertion, presumably at attB. Thus, the replacement reduced but did not prevent reinsertion at the original site. We surmise that a partial deletion of host DNA could increase insertion by appropriately repositioning the P' arm- and core-type Int binding sites and that such an event could be a step in the evolution of a new insertion specificity.

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TABLE 2. Int-promoted insertion of attR ( bio936) into strain RW4209 (MG1655 306[srl-recA])a

An unexpected outcome of this work is the excess of excisive relative to integrative recombination of wild-type sites catalyzed by N99D-E319R. The bias depends on the substrates, since it is much reduced for the sraF sites (Table 1). The converse bias is detected in the quintuple mutant Int-catalyzed sraF recombinations. The relatively low level of excision is also substrate dependent since the wild-type HK022 substrate is excised at high frequency by this Int (Table 1). Integration and excision differ in many ways: requirement for accessory proteins, formation of the recombinogenic intasomes, usage of arm-type recognition sites, etc. Our findings indicate that information in the core sites is not equally used during the two reactions.

 
* Corresponding author. Present address: Biocenter Ltd., Temesvári krt. 62, Szeged 6726, Hungary. Phone: 36 30 567 8941. Fax: 36 62 599 751. E-mail: dorgai{at}biocenter.hu.

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1. Azaro, M. A., and A. Landy. 2002. Integrase and the Int family, p. 118-148. In N. L. Craig, R. Craigie, M. Gellert, and A. M. Lambowitz (ed.), Mobile DNA II. ASM Press, Washington D.C.
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2. Azaro, M. A., and A. Landy. 1997. The isomeric preference of Holliday junctions influences resolution bias by lambda integrase. EMBO J. 16:3744-3755.[CrossRef][Medline]
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3. Dorgai, L., E. Yagil, and R. A. Weisberg. 1995. Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases. J. Mol. Biol. 252:178-188.[CrossRef][Medline]
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4. Dorgai, L., S. Sloan, and R. A. Weisberg. 1998. Recognition of core binding sites by bacteriophage integrases. J. Mol. Biol. 277:1059-1070.[CrossRef][Medline]
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5. Rutkai, E., L. Dorgai, R. Sirot, E. Yagil, and R. A. Weisberg. 2003. Analysis of insertion into secondary attachment sites by phage , and by Int mutants with altered recombination specificity. J. Mol. Biol. 329:983-996.[Medline]
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6. Weisberg, R. A., L. W. Enquist, C. Foeller, and A. Landy. 1983. Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda. J. Mol. Biol. 170:319-342.[CrossRef][Medline]

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Journal of Bacteriology, May 2006, p. 3409-3411, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3409-3411.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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