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Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn, directs the relative trajectories of the CC motifs on each subunit of the att -bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal length CC motifs and 14 residues surrounding the tip where pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggests that molecular interactions between CC motif tips may differ in integrative ( attP x attB ) and excisive ( attL x attR ) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminantly, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after initial assembly of the integrase synaptic tetramer. Importance The robust and exquisitely-regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage, and in the case of the A118 prophage, are an important virulence factor by Listeria monocytogenes . The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

IS607-family transposons are unusual because they do not have terminal inverted repeats or generate target site duplications. They encode two protein-coding genes, but only tnpA is required for transposition. Our X-ray structures confirm that TnpA is a member of the serine recombinase (SR) family, but the chemically-inactive quaternary structure of the dimer, along with the N-terminal location of the DNA binding domain, are different from other SRs. TnpA dimers from IS1535 cooperatively associate with multiple subterminal repeats, which together with additional nonspecific binding, form a nucleoprotein filament on one transposon end that efficiently captures a second unbound end to generate the paired-end complex (PEC). Formation of the PEC does not require a change in the dimeric structure of the catalytic domain, but remodeling of the C-terminal α-helical region is involved. We posit that the PEC recruits a chemically-active conformer of TnpA to the transposon end to initiate DNA chemistry.

IS607-family transposons are unusual because they do not have terminal inverted repeats or generate target site duplications. They encode two protein-coding genes, but only tnpA is required for transposition. Our X-ray structures confirm that TnpA is a member of the serine recombinase (SR) family, but the chemically-inactive quaternary structure of the dimer, along with the N-terminal location of the DNA binding domain, are different from other SRs. TnpA dimers from IS1535 cooperatively associate with multiple subterminal repeats, which together with additional nonspecific binding, form a nucleoprotein filament on one transposon end that efficiently captures a second unbound end to generate the paired-end complex (PEC). Formation of the PEC does not require a change in the dimeric structure of the catalytic domain, but remodeling of the C-terminal α-helical region is involved. We posit that the PEC recruits a chemically-active conformer of TnpA to the transposon end to initiate DNA chemistry.

Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI: http://dx.doi.org/10.7554/eLife.01211.001, eLife digest Many processes in biology rely on enzymes that break both the strands in a DNA molecule, then rearrange the strands, and finally join them back together in a new configuration. These recombination reactions can, for example, change the positions of genetic elements such as enhancers and promoters within the DNA molecule and, therefore, influence how a given gene is expressed as a protein. Cells need to be able to control recombination reactions because they can lead to leukemia and lymphomas if they go wrong. The enzymes that catalyze these recombination reactions are called recombinases. One type of recombinase binds to specific sequences of DNA bases and uses an amino acid in the enzyme–usually serine or tyrosine–to break and rejoin the DNA strands. Recombination reactions require the assembly of complexes containing many proteins bound to DNA. Tyrosine recombinases form relatively simple protein-DNA complexes, and these have been studied in detail. Serine recombinases, on the other hand, form more elaborate protein-DNA complexes, and much less is known about these. Now McLean et al. have unraveled the mechanism that a serine recombinase called Hin uses to reverse the direction of a stretch of chromosomal DNA in the bacteria Salmonella enterica. Inverting this stretch of DNA–which contains about 1000 base pairs–changes the position of a gene promoter that is responsible for the production of flagellin, which is the protein that enables the bacterium to move. This is one of the tricks that Salmonella uses to evade the immune system of its host. Previous research has established that four Hin subunits and two copies of a protein called Fis are needed to invert this stretch of DNA: two Hin subunits bind to each of the two hix recombination sites, and the Fis proteins (which are dimers) bind to each end of an enhancer that is located between the hix sites. A protein called HU then causes the DNA to bend and form a loop, and the four Hin subunits and the two Fis dimers all come together at the enhancer to form a structure called the invertasome where the recombination reaction occurs. All four DNA strands at the crossover point are broken as a result of a near simultaneous attack by the catalytic serine amino acids in the Hin subunits. One pair of Hin subunits–and the two DNA strands attached to them–then rotate by 180 degrees around the other pair of Hin subunits. This means that the stretch of DNA between the hix sites is inverted when the DNA strands are rejoined at the end of the reaction. Enhancers often regulate transcription and other reactions from a distance. McLean et al. reveal how an enhancer of a DNA recombination reaction works. The pairs of Hin subunits that initially bind to the DNA are not catalytically active, but when they are brought together by the enhancer and form a tetramer, they become active. Two of the Hin subunits are clamped onto the enhancer by the Fis dimers and by directly interacting with the enhancer DNA, but the other two (and the DNA strands attached to them) are free to rotate within the tetramer. In the Salmonella chromosome the enhancer is located close to one of the hix sites (∼100 base pairs away from it), so the length of the DNA between the enhancer and hix site physically limits the number of Hin subunit rotations to just one. DOI: http://dx.doi.org/10.7554/eLife.01211.002

This study describes the genome of temperate Siphoviridae phage DW2, which is routinely propagated on Staphylococcus aureus DPC5246. The 41941 bp genome revealed an open reading frame (ORF1) which has a high level of homology with members of the resolvase subfamily of site-specific serine recombinase, involved in chromosomal integration and excision. In contrast, the majority of staphylococcal phages reported to date encode tyrosine recombinases. Two putative genes encoded by phage DW2 (ORF15 and ORF24) were highly homologous to the NWMN0273 and NWMN0280 genes encoding virulence factors carried on the genome of ϕNM4, a prophage in the genome of S. aureus Newman. Phage DW2 also encodes proteins highly homologous to two well-characterized Staphylococcus aureus pathogenicity island derepressors encoded by the staphylococcal helper phage 80α indicating that it may similarly act as a helper phage for mobility of pathogenicity islands in S. aureus. This study also focused on the enzybiotic potential of phage DW2. The structure of the putative endolysin and tail hydrolase were investigated and used as the basis for a cloning strategy to create recombinant peptidoglycan hydrolyzing proteins. After overexpression in E. coli, four of these proteins (LysDW2, THDW2, CHAPE1-153, and CHAPE1-163) were demonstrated to have hydrolytic activity against peptidoglycan of S. aureus and thus represent novel candidates for exploitation as enzybiotics.