Making serine integrases work for us

DNA site-specific recombinases are enzymes (often associated with mobile DNA elements) that catalyse breaking and rejoining of DNA strands at specific points, thereby bringing about precise genetic rearrangements. Serine integrases are a group of recombinases derived from bacteriophages. Their unusual properties, including directionality of recombination and simple site requirements, are leading to their development as efficient, versatile tools for applications in experimental biology, biotechnology, synthetic biology and gene therapy. This article summarizes our current knowledge of serine integrase structure and mechanism, then outlines key factors that affect the performance of these phage recombination systems. Recently published studies, that have expanded the repertoire of available systems and reveal system-specific characteristics, will help us to choose the best integrases for envisaged applications

A Family of Developmentally Excised DNA Elements in \u3cem\u3eTetrahymena\u3c/em\u3e is under Selective Pressure to Maintain an Open Reading Frame Encoding an Integrase-Like Protein

Tlr1 is a member of a family of ~20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan Tetrahymena. Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen TIC transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the Tetrahymena genome, where TAA and TAG encode Gin. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed

Application of Serine Integrases for Secondary Metabolite Pathway Assembly in Streptomyces

Serine integrases have been shown to be efficient tools for metabolic pathway assembly. To further improve the flexibility and efficiency of pathway engineering via serine integrases, we explored how multiple orthogonally active serine integrases can be applied for use in vitro for the heterologous expression of complex biosynthesis pathways in Streptomyces spp., the major producers of useful bioactive natural products. The results show that multiple orthogonal serine integrases efficiently assemble the genes from a complex biosynthesis pathway in a single in vitro recombination reaction, potentially permitting a versatile combinatorial assembly approach. Furthermore, the assembly strategy also permitted the incorporation of a well-characterised promoter upstream of each gene for expression in a heterologous host. The results demonstrate how site-specific recombination based on orthogonal serine integrases can be applied in Streptomyces spp

Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae

Background: Phage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology. Results: The activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination. Conclusions: The results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wβ and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

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

Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N2-fixing root nodules on diverse and globally distributed angiosperms in the “actinorhizal” symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%–98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses

New Applications for Phage Integrases

Within the last twenty-five years bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide-range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimisation, bio-computing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line

A proposed mechanism for IS607-family serine transposases

Background The transposases encoded by the IS607 family of mobile elements are unusual serine recombinases with an inverted domain order and minimal specificity for target DNA.<p></p> Results Structural genomics groups have determined three crystal structures of the catalytic domains of IS607 family transposases. The dimers formed by these catalytic domains are very different from those seen for other serine recombinases and include interactions that usually only occur upon formation of a synaptic tetramer.<p></p> Conclusions Based on these structures, we propose a model for how IS607-family transposases could form a synaptic tetramer. The model suggests that, unlike other serine recombinases, these enzymes carry out sequence-specific DNA binding and catalysis in trans: the DNA binding and catalytic domains of each subunit are proposed to interact with different DNA duplexes. The model also suggests an explanation for the minimal target DNA specificity.<p></p&gt