Measurements of DNA-loop formation via Cre-mediated recombination

Shoura, Massa J.; Vetcher, Alexandre A.; Giovan, Stefan M.; Bardai, Farah H.; Bharadwaj, Anusha; Kesinger, Matthew R.; Levene, Stephen D.

doi:10.1093/nar/gks430

Cited by 22 publications

(46 citation statements)

References 51 publications

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“…Single-exponential fits give rates of 0.01 min −1 and 0.96 min −1 for native and SDS-stopped reactions, respectively, indicating that the lifetime of the TS-P complex is ∼75 min. Our dissociation rates agree with those derived from a kinetic model of Cre recombination (18) and previous observations of highly stable complexes (26,27).…”

Section: Resultssupporting

confidence: 89%

“…After completion of recombination, we observed long-lived recombinant product synaptic complexes (TS-P) refractory to subsequent recombination. This lifetime agrees with ensemble measurements (26,27) but is significantly longer than observed in a TPM study (12); however, we note that the use of a 200-nm bead in the TPM work generates an effective stretching force on the DNA (29) and may have increased the rate of complex dissociation. Previous ensemble biochemical data had measured similar dissociation constants (K d ) for the synaptic complexes of wt Cre and K201A (14 and 9 nM, respectively; ref 21).…”

Section: Rate-limitingsupporting

confidence: 87%

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Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Pinkney

Zawadzki

Mazuryk

et al. 2012

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

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Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large-and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.tethered fluorophore motion | site-specific DNA recombination | Holliday junction dynamics | alternating laser excitation | protein-DNA interactions S ite-specific DNA recombination is the protein-mediated cleavage, exchange, and rejoining of DNA strands between two duplexes containing a specific sequence. Tyrosine recombinases are involved in the integration and excision of viral genomes, resolution of chromosome dimers, and gene expression (1). This large family of proteins shares a general mechanism of recombination whereby a tetramer of recombinases mediates sequential pairs of strand exchange between two DNA duplexes; the first pair forms a Holliday junction (HJ) intermediate, whereas the second leads to resolution of the HJ to recombinant product (Fig. 1A). DNA cleavage requires no external energy factors, as the bond energy is stored during strand exchange as a covalent protein-DNA linkage.One of the best studied tyrosine recombinases is Cre, a 38 kDa protein of the bacteriophage P1 of Escherichia coli. Cre catalyzes cyclization of the viral genome and resolution of plasmid dimers during replication (2). Cre mediates recombination between 34 bp DNA sequences named loxP (Fig. 1B). Unlike other recombinases [e.g., XerCD (3) and λ Int (4)], Cre does not require accessory...

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Section: Resultssupporting

confidence: 89%

Section: Rate-limitingsupporting

confidence: 87%

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Pinkney

Zawadzki

Mazuryk

et al. 2012

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

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“…Our 900-bp estimate of j LOOP = 54 nM is similar to the j LOOP = 37 nM obtained for an 870-bp loop formed by Cre recombinase (15). At the 3,200-bp spacing, our in vitro j LOOP fell to ∼6 nM, which is comparable to 18 nM at 3,044 bp for Cre (15) and ∼10 nM for DNA cyclization at 4,000 bp (14). TPM analysis of CI looping at 2,300 bp gave j LOOP = 24 nM (17), somewhat higher than our TPM values (Fig.…”

Section: Effect Of Separation On Long-range Looping By λ Repressor Insupporting

confidence: 80%

“…The TPM results of Johnson et al (26) indicate even poorer looping with short spacers, with j LOOP values of 0.3-4 nM for different ∼100-bp LacI loops. Our 900-bp estimate of j LOOP = 54 nM is similar to the j LOOP = 37 nM obtained for an 870-bp loop formed by Cre recombinase (15). At the 3,200-bp spacing, our in vitro j LOOP fell to ∼6 nM, which is comparable to 18 nM at 3,044 bp for Cre (15) and ∼10 nM for DNA cyclization at 4,000 bp (14).…”

Section: Effect Of Separation On Long-range Looping By λ Repressor Insupporting

confidence: 80%

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Priest

Cui

Kumar

et al. 2013

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

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Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to longrange DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250-to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5-to 10-kb distances, was able to increase contact probability 10-to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5-to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells. j factor | TPM | promoter-enhancer I nteractions between proteins bound to separate sites on the same DNA molecule are critical in gene regulation and other DNA processes (1-4). The DNA separation between functionally interacting sites ranges from a few base pairs to hundreds of kilobase pairs, as in the case of some eukaryotic enhancers and their promoters. At short separations, it is clear that the DNA acts as a tether that keeps one site in the vicinity of the other so that the proteins at one site can find the other site in 3D space more efficiently than if they were free in solution (Fig. 1). Tethering is also a way to provide specificity because it aids interaction with linked sites but not unlinked sites. However, as the separation between the sites increases, this tethering effect becomes weaker, and it is not understood how the DNA linkage between widely separated sites, for example, between enhancers and promoters, provides the efficiency and specificity required for proper regulation.The effect of DNA tethering can be quantified by the factor j LOOP (M), the effective molar concentration of one site on the DNA relative to the other, or as the free energy of the DNA looping reaction ΔG LOOP (Fig. 1) (5-9). These parameters are interconvertible: ΔG LOOP = -RTlnj LOOP (kcal/mol; the reference j is 1 M). The formation of a naked DNA loop is in itself an energetically unfavorable reaction (ΔG LOOP is positive) under physiological conditions due to the enthalpic cost of DNA bending and twisting (particularly important for s...

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“…Plasmids and synthetic DNA mini-circles As a template for generating reference DNA circles, we used a plasmid with two directly repeated loxP sites cloned in the backbone of the generic vector pGEM5Zf(+). (Shoura and Levene; Shoura et al) The resulting plasmid, pCS2DloxPzero (Shoura et al, 2012) allows insertion of arbitrary spacer sequences (gBlocks, IDT) between the two loxP sites by linearizing the plasmid with both NotI and PstI (NEB). Using pCS2DloxPZero as a vector, we cloned a 378 bp insert sequence between the two loxP sites, resulting in plasmid pCS2DloxP378.…”

Section: Library Prep and Low-input Nextera Protocolmentioning

confidence: 99%

Beyond the Linear Genome: Comprehensive Determination of the Endogenous Circular Elements inC. elegansand Human Genomes via an Unbiased Genomic-Biophysical Method

Shoura

Gabdank

Hansen

et al. 2017

Preprint

Self Cite

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Investigations aimed at defining the 3-D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. High-throughput sequencing has only recently made extensive genomic mapping of eccDNA sequences possible and had yet to be applied using a rigorous approach that distinguishes ascertainment bias from true enrichment. Here, we define eccDNA distribution, utilizing a set of unbiased topologydependent approaches for enrichment and characterization. We use parallel biophysical, enzymatic, and informatic approaches to obtain a comprehensive profiling of eccDNA in C. elegans and in three human cell types, where eccDNAs were previously uncharacterized. We also provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture in which endogenous genomic DNA circles are present in normal physiological DNA metabolism, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

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Measurements of DNA-loop formation via Cre-mediated recombination

Cited by 22 publications

References 51 publications

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Beyond the Linear Genome: Comprehensive Determination of the Endogenous Circular Elements inC. elegansand Human Genomes via an Unbiased Genomic-Biophysical Method

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