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Abstract: Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs. [Copyright &y& Elsevier]

Abstract: Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described. [Copyright &y& Elsevier]

Abstract: Site-specific recombination on supercoiled circular DNA molecules can yield a variety of knots and catenanes. Twist knots are some of the most common conformations of these products, and they can act as substrates for further rounds of site-specific recombination. They are also one of the simplest families of knots and catenanes. Yet, our systematic understanding of their implication in DNA and important cellular processes such as site-specific recombination is very limited. Here, we present a topological model of site-specific recombination characterizing all possible products of this reaction on twist knot substrates, extending the previous work of Buck and Flapan. We illustrate how to use our model to examine previously uncharacterized experimental data. We also show how our model can help determine the sequence of products in multiple rounds of processive recombination and distinguish between products of processive and distributive recombinations. This model studies generic site-specific recombination on arbitrary twist knot substrates, a subject for which there is limited global understanding. We also provide a systematic method of applying our model to a variety of different recombination systems. [Copyright &y& Elsevier]

Abstract: Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3′-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction. [Copyright &y& Elsevier]