Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET

Rutkauskas, Danielis; Petkelytė, Milda; Naujalis, Paulius; Sasnauskas, Giedrius; Тамулайтис, Г.; Zaremba, Mindaugas; Šikšnys, Virginijus

doi:10.1021/jp504546v

Cited by 8 publications

(22 citation statements)

References 41 publications

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“…The Cy5 label is close to the DNA end distal from the surface and is, therefore, on a tether a few hundreds of nanometers long and can diffuse within the range permitted by the tether. The occurrence of FRET for this construct was previously observed in ensemble experiments (22).…”

Section: Resultssupporting

confidence: 72%

“…1 A). The proper placement of the dye labels on DNA was determined from the crystal structure of the DNA-Ecl18kI complex (22,29) by using the tool for FRET-restrained positioning and screening (30) to simulate the distances between the geometrically accessible volumes (AV) of the two dyes. No FRET is expected in a ''parallel'' loop configuration where the interdye distance is above the range accessible to FRET (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The scheme for the fragment synthesis was the same as described previously (22), and the specific details are provided in the Supporting Material. In short, FRETpair-labeled DNA fragments were synthesized using labeled primers in the PCR.…”

Section: Protein and Dna Fragmentsmentioning

confidence: 99%

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DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Tutkus

Marčiulionis

Sasnauskas

et al. 2017

Biophysical Journal

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The single-molecule Fö rster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field. In combination with FRET it allowed us to reveal and characterize the dynamics of what had been inaccessible conformations of the DNA-protein complex. Our model system, restriction enzyme Ecl18kI, interacts with a FRET pair-labeled DNA fragment to form two different DNA loop conformations. The DNA-protein interaction geometry is such that the efficient FRET is expected for one of these conformations-''antiparallel'' loop. In the alternative ''parallel'' loop, the expected distance between the dyes is outside the range accessible by FRET. Therefore, ''antiparallel'' looping is observed in a single-molecule time trajectory as discrete transitions to a state of high FRET efficiency. At the same time, transitions to a high-intensity state of the directly excited acceptor fluorophore on a DNA tether are due to a change of its average position in the evanescent field of excitation and can be associated with a loop of either ''parallel'' or ''antiparallel'' configuration. Simultaneous analysis of FRET and acceptor intensity trajectories then allows us to discriminate different DNA loop conformations and access the average lifetimes of different states.

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

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DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Tutkus

Marčiulionis

Sasnauskas

et al. 2017

Biophysical Journal

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“…The topic of the present work is quantitative characterization of the real‐time dynamics of interaction between DNA and a well‐characterized homotetrameric type IIF REase—NgoMIV, capable of simultaneous binding of two 5′‐GCCGGC‐3′ target sites via two identical DNA‐binding surfaces. Since NgoMIV tetramer contains two DNA‐binding clefts, compared to Ecl18kI it presents a more complex example of interactions with a two‐target DNA described by a branched kinetic scheme. NgoMIV‐induced DNA looping has been studied previously by measuring inhibition of Tn21 resolvase reaction, but the loops were found to be too unstable to be characterized by this method .…”

Section: Introductionmentioning

confidence: 54%

“…By combining these techniques, we were able to determine the rate constants for DNA unlooping and protein dissociation in the kinetic scheme of NgoMIV ‐ DNA interactions. The dynamics of NgoMIV interaction with DNA proved to be highly heterogeneous exhibiting broadly different loop stabilities—the feature that was not detected in our previous work on DNA interactions with dimeric REase Ecl18kI . The observed diversity is attributed to the different forms of NgoMIV‐induced DNA loops connected with the fact that NgoMIV is a tetrameric protein and also to the conformational heterogeneity of individual NgoMIV molecules.…”

Section: Introductionmentioning

confidence: 99%

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

2017

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Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.

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smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease

et al. 2023

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Protein–DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans. Type IIS restriction endonucleases rely on such interactions. They play a crucial role in safeguarding bacteria against foreign DNA, including viral genetic material. BfiI, a type IIS restriction endonuclease, acts upon a specific asymmetric sequence, 5-ACTGGG-3, and precisely cuts both upper and lower DNA strands at fixed locations downstream of this sequence. Here, we present two single-molecule Förster resonance energy-transfer-based assays to study such interactions in a BfiI–DNA system. The first assay focuses on DNA looping, detecting both “Phi”- and “U”-shaped DNA looping events. The second assay only allows in trans BfiI–target DNA interactions, improving the specificity and reducing the limits on observation time. With total internal reflection fluorescence microscopy, we directly observe on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binds longer to target sites and that BfiI rarely changes conformations during binding. This newly developed assay could be employed for other DNA-interacting proteins that bind two targets and for the dsDNA substrate BfiI-PAINT, a useful strategy for DNA stretch assays and other super-resolution fluorescence microscopy studies.

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Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET

Cited by 8 publications

References 41 publications

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease

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