Synapsis and catalysis by activated Tn3 resolvase mutants

Olorunniji, Femi J.; He, Jin; Wenwieser, Sandra V. C. T.; Boocock, Martin R.; Stark, W. Marshall

doi:10.1093/nar/gkn885

Cited by 36 publications

(80 citation statements)

References 40 publications

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“…In their natural context, small serine recombinases, such as the transposon resolvases and DNA invertases, stop after one round of strand exchange (1,25,26), even though the catalytic module (comprising two crossover sites synapsed by a recombinase tetramer) is fully competent for indefinite rounds of rotation (27). This selectivity is imposed by regulatory components involving accessory DNA sites and proteins (see introductory paragraphs).…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

“…Aliquots from the reaction samples (20 μL) were treated with restriction enzymes or DNase I as described (15,27). Loading buffer [25 mM Tris·HCl (pH 8.2), 20% (wt/vol) Ficoll, 0.5% sodium dodecyl sulphate, 5 mg/ml protease K, 0.25 mg/ml bromophenol blue; 5 μl] was added to each sample.…”

Section: Methodsmentioning

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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“…It binds to DNA double-strand breaks to form a synapsis and recruits other proteins such as the ligase complex (17)(18)(19)(20). As a central event of transposition, PEC formation is also characterized by a synapsis between both ends of the transposon (21,22), and we thus addressed the question of whether PBase interacting with DNA-PK contributes to PEC formation.…”

Section: Dna-pk Expression Significantly Impacts On the Transpositionmentioning

confidence: 99%

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Jin

Chen

Zhao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.piggyBac | DNA-PK | paired-end complex formation | transposition efficiency | CE transposon vectors T ransposition allows DNA transposons to serve as powerful genetic manipulation tools. During transposition, DNA transposons follow a "cut-and-paste" manner. A paired-end complex (PEC) is first formed by aligning both ends of the transposon, which is then excised and inserted into new loci (1, 2). Although the transposon-encoded transposase is suggested to be sufficient for PEC formation, it is unlikely to be the only protein involved in transposition. In fact, many DNA transposons are nonfunctional outside their natural hosts, suggesting the effects of host factors (3).piggyBac (PB) is a DNA transposon originally identified from the cabbage looper moth (4). Modified PB is highly active in mouse and human cells (5). Because of its potent transposition efficiency and ability to carry large insertions, PB provides unique opportunities for mutagenesis and transgenesis in mammalian systems (6-10). Although the PB transposon has a wide host range, including mammals, its transposition efficiency varies in cultured cells from different mammalian species (7,9). Thus, host factors may still be involved in the proces...

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“…10.4a ). Furthermore, recombinase mutants are known that do not require accessory cofactors for recombination activity (Burke et al 2004 ;Klippel et al 1988 ;Olorunniji et al 2008 ) . Akopian et al extended the studies of Schneider et al 2000 by replacing the DNA-binding domain of the Tn3 resolvase with the zinc fi nger DNA-recognition domain of the natural mouse transcription factor Zif268 (Akopian et al 2003 ) .…”

Section: Retargeting Modular Recombinases With New Dna-binding Domainsmentioning

confidence: 99%

Targeted Plasmid Integration into the Human Genome by Engineered Recombinases

Gersbach

Barbas

2012

Site-Directed Insertion of Transgenes

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The targeted integration of transgenes into cellular genomes is central to numerous applications in biotechnology, basic science, and medicine. In recent years, a variety of advances have improved upon conventional methods for site-speci fi c transgene integration. Most of these methods involve nucleases that cleave DNA to activate DNA repair pathways including homologous recombination or integrases that fully catalyze the integration reaction but are limited in their capacity to target new sites in the genome. Recently, zinc-fi nger recombinases have emerged as a class of engineered enzymes that combines the strengths of both of these previous methods. Zinc-fi nger recombinases can fully and autonomously catalyze plasmid integration into the genome of mammalian cells without creating free DNA breaks. In addition, they can be engineered to target new genomic recognition sites by exchanging the modular and programmable DNA-binding domain and through directed evolution of the serine recombinase catalytic domain. This chapter reviews the development of the zinc-fi nger recombinase technology, including discussions of its strengths and weaknesses and the future directions necessary to translate this technology into routine use for transgene integration into cellular genomes.

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Synapsis and catalysis by activated Tn3 resolvase mutants

Cited by 36 publications

References 40 publications

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Targeted Plasmid Integration into the Human Genome by Engineered Recombinases

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