Cre Recombinase

Duyne, Gregory D. Van

doi:10.1128/9781555819217.ch5

Cited by 34 publications

(70 citation statements)

References 119 publications

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“…Site specific DNA recombinases represent an attractive option for genome engineeringthe insertion or exchange of genes into precise locations in chromosomes [1][2][3][4] . The tyrosine recombinase family of phage-derived enzymes (e.g., λ-integrase, Cre and Flp recombinases), which evolved to facilitate viral infection, gene transposition and bacterial pathogenesis, have proven useful for applications that include DNA subcloning without restriction enzymes, and conditional expression of target genes [5][6][7] . These proteins bind specifically to pairs of inverted short palindromic DNA sequences (recombinase binding elements; RBEs) and mediate recombination by assembly of tetrameric intasomes (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…These proteins bind specifically to pairs of inverted short palindromic DNA sequences (recombinase binding elements; RBEs) and mediate recombination by assembly of tetrameric intasomes (Fig. 1A and B) which perform concerted DNA strand cleavage, exchange and ligation reactions 5,8,9 . Compared to other genome engineering approaches 10,11 , site specific DNA recombinases have the advantage that they allow a high degree of specificity, don't require involvement of additional host-encoded factors, and are capable of generating cleanly integrated double-stranded DNA products 3,[12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

“…Cre (Causes Recombination) is the best studied member of the tyrosine recombinase family, yet fundamental gaps exist in our understanding of its mechanism of DNA site selection, intasome assembly, and allosteric control of DNA cleavage. The overall mechanism of Cremediated DNA recombination has been mapped out via many biochemical and crystallographic studies 5,[15][16][17][18][19] . The enzyme catalyzes recombination between a pair of homologous 34 bp loxP sites (Fig.…”

Section: Introductionmentioning

confidence: 99%

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DNA Binding Induces acistotransSwitch in Cre Recombinase to Enable Intasome Assembly

Unnikrishnan

Amero

Yadav

et al. 2020

Preprint

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Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (Cre NTD , Cre Cat ) to be structurally independent. Amide 15 N relaxation measurements of the Cre Cat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the Cterminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre.Chemical shift perturbations and intra-and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site.Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage.Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

DNA Binding Induces acistotransSwitch in Cre Recombinase to Enable Intasome Assembly

Unnikrishnan

Amero

Yadav

et al. 2020

Preprint

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“…17,18 In the life cycle of phage P1 Cre plays a critical role in genome maintenance by unlinking newly replicated sister chromosomes to ensure their proper segregation following DNA replication. 19 Cre acts at a 34-bp wild-type DNA 3 recombination-target sequence called loxP, which consists of 13-bp perfect inverted repeats flanking a non-palindromic 8-bp core region 20 and defines the site's absolute orientation. On a linear DNA molecule Cre-mediated recombination involving a pair of tandemly repeated loxP sequences generates a pair of deletion products (one linear, one circular), each bearing a single copy of loxP (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…21 As with other members of the λ-int superfamily, recombination takes place via two successive rounds of DNA-strand cleavage and exchange steps that respectively generate and resolve a four-stranded, Holliday-junction (HJ) DNA intermediate. 20,22 High-resolution crystal structures of Cre synaptic complexes formed with duplex and junction DNAs provided the first insights into mechanistic details of recombination in these systems. On the basis of these structures, the chemical steps in Cre recombination have been explained in terms of DNA strand exchanges taking place within a twofold-symmetric, nearly square-planar arrangement of the DNA duplexes.…”

Section: Introductionmentioning

confidence: 99%

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Shoura

Giovan

Vetcher

et al. 2019

Preprint

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In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of DNA target sites. The strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of the DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. Here we use the kinetics of loop closure involving closely spaced (131-151 bp), directly repeated loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-ofplane HJ distortion. This result unequivocally stands in contrast to the square-planar intermediate geometry determined from crystallographic data for the Cre-loxP system and other int-superfamily recombinases. J measurements carried out with an isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex may result from the temporal averaging of diverse right-handed and achiral structures. Applied to Cre recombinase, and other biological systems, our approach bridges the static pictures provided by crystal structures and the natural dynamics of macromolecules in vivo. This approach thus advances a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

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Genome Editing for the Production of Natural Products in Escherichia coli

Zebec

Scrutton

2018

Adv. Biosys.

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Natural products such as secondary metabolites (e.g., plant terpenoids) are found to be a major source of bioactive compounds. These natural products accumulate as complex mixtures with other related compounds and this chemical complexity adds cost to the downstream recovery and purification of natural products from plant biomass. One aim of synthetic biology and metabolic engineering programmes is to produce such compounds from synthetic gene clusters in heterologous hosts and thereby achieve more targeted and affordable production. Both fungi and bacteria are common hosts for metabolic engineering in industry. Fungal hosts include Penicillium chrysogenum, Saccharomyces cerevisiae, Aspergillus niger and the bacterial hosts Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. E. coli is often selected as a host given the ease of its genetic manipulation and the long history of using this organism in laboratory‐based bioengineering. The bioengineering of E. coli extends also to feedstock pathways to interface and optimize the production of high value compounds from widely available and inexpensive carbon sources. Genome editing is important in these microbial bioengineering programmes and is needed to isolate stable strains and to optimize production. Herein, this review discusses frequently used methods for genome editing in E. coli in relation to the production of natural compounds and chemicals.

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Cre Recombinase

Cited by 34 publications

References 119 publications

DNA Binding Induces acistotransSwitch in Cre Recombinase to Enable Intasome Assembly

DNA Binding Induces acistotransSwitch in Cre Recombinase to Enable Intasome Assembly

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Genome Editing for the Production of Natural Products in Escherichia coli

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