Site‐Specific DNA Recombinases as Instruments for Genomic Surgery

Akopian, Aram; Stark, W. Marshall

doi:10.1016/s0065-2660(05)55001-6

Cited by 38 publications

(42 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Site-specific recombination reactions are ideally suited to singlemolecule analysis because they involve discrete complexes that undergo predictably orchestrated catalytic steps. A range of important DNA rearrangements in prokaryotes and simple eukaryotes are mediated by site-specific recombination, which is also widely exploited as a tool in genome engineering (1)(2)(3). Tyrosine family recombinases facilitate the propagation of plasmids and viruses, and have key roles in plasmid and chromosome stability and in the horizontal transfer of pathogenicity and antibiotic resistance determinants.…”

mentioning

confidence: 99%

Conformational transitions during FtsK translocase activation of individual XerCD– dif recombination complexes

Zawadzki

May

Baker

et al. 2013

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

View full text Add to dashboard Cite

Three single-molecule techniques have been used simultaneously and in tandem to track the formation in vitro of single XerCD-dif recombination complexes. We observed the arrival of the FtsK translocase at individual preformed synaptic complexes and demonstrated the conformational change that occurs during their activation. We then followed the reaction intermediate transitions as Holliday junctions formed through catalysis by XerD, isomerized, and were converted by XerC to reaction products, which then dissociated. These observations, along with the calculated intermediate lifetimes, inform the reaction mechanism, which plays a key role in chromosome unlinking in most bacteria with circular chromosomes.tethered fluorophore motion | site-specific DNA recombination | protein-DNA interaction | chromosome segregation | single-molecule FRET

show abstract

mentioning

confidence: 99%

Conformational transitions during FtsK translocase activation of individual XerCD– dif recombination complexes

Zawadzki

May

Baker

et al. 2013

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

View full text Add to dashboard Cite

show abstract

“…Recently, site-specific recombinases have emerged as powerful tools for advanced genome engineering (2,3). The exquisite sequence specificities of recombination systems such as Cre/lox, FLP/FRT, and φC31/ att allow researchers to accurately modify genetic information for a variety of applications (4)(5)(6).…”

mentioning

confidence: 99%

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Gaj

Mercer

Gersbach

et al. 2010

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

View full text Add to dashboard Cite

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.gene targeting | protein engineering | site-specific recombination | zinc-finger recombinase S ite-specific recombinases are essential for a variety of diverse biological processes, including the integration and excision of viral genomes, the transposition of mobile genetic elements, and the regulation of gene expression (1). Recently, site-specific recombinases have emerged as powerful tools for advanced genome engineering (2, 3). The exquisite sequence specificities of recombination systems such as Cre/lox, FLP/FRT, and φC31/ att allow researchers to accurately modify genetic information for a variety of applications (4-6). However, DNA sequence constraints imposed by site-specific recombinases make routine modification of cellular genomes contingent on the presence of artificially introduced recognition sequences. As a result, a number of attempts have been made to circumvent or reprogram the strict DNA sequence specificities observed in these systems (7-9). Despite these efforts, engineered site-specific recombinase variants often exhibit considerably relaxed DNA sequence specificities (8, 10-14), a detrimental byproduct that often results in adverse off-target chromosomal modification (15-17). Thus, there is significant interest in the development of generalized protein engineering strategies capable...

show abstract

“…21,22 Moreover, during recent years a massive effort has been put into the development of Flp-or Crebased gene integration systems for therapeutic purposes. [23][24][25][26] At present there exist no quick easy-to-handle activity assays with a sensitivity high enough to reliably detect Flp and/or Cre activity in small crude biological samples. Hence, the presented multiplexed singlemolecule RCA-based detection of Flp and Cre activities, requiring only the access to a fluorescence microscope, are of anticipated value for both scientific and future clinical applications as a positive control for protocols relying on recombinase activity.…”

mentioning

confidence: 99%

Multiplexed Detection of Site Specific Recombinase and DNA Topoisomerase Activities at the Single Molecule Level

et al. 2009

View full text Add to dashboard Cite

We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.

show abstract

Site‐Specific DNA Recombinases as Instruments for Genomic Surgery

Cited by 38 publications

References 73 publications

Conformational transitions during FtsK translocase activation of individual XerCD– dif recombination complexes

Conformational transitions during FtsK translocase activation of individual XerCD– dif recombination complexes

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Multiplexed Detection of Site Specific Recombinase and DNA Topoisomerase Activities at the Single Molecule Level

Contact Info

Product

Resources

About