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

Abstract

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.

Fixed DNA Molecule Arrays for High-Throughput Single DNA–Protein Interaction Studies

“…Then, it was purified using the GeneJET PCR Purification Kit (ThermoFisher Scientific). Biotinylated 350 bp long dsDNA with the Atto647N dye was prepared using a two-step PCR reaction and ligation with the biotinylated DNA leg in the same way as described previously . The protocol is presented in detail in the Supporting Information file.…”

Advancing Understanding of DNA-BfiI Restriction Endonuclease Cis and Trans Interactions through smFRET Technology

“…1B). 28 In our study, both DNA loop shapes resulted in non-zero FRET efficiency. However, NgoMIV limits our assay, since increased protein concentration increases the probability of having two tetramers per single DNA fragment containing two target sites.…”

Prokaryotic Argonaute From Archaeoglobus Fulgidus Interacts With DNA as a Homodimer

“…In the rst scenario, the reaction may proceed via (i) association of free DNA (species "0") with a single WT AfAgo dimer, which binds to one DNA end (species "1"); (ii) capture of the second DNA terminus by the DNA-bound AfAgo dimer in an intramolecular reaction, resulting in a looped complex (species "2"); (iii) alternatively, association of the second WT AfAgo dimer with the unoccupied second DNA end of species "1" leads to species "3", which is no longer capable of loop formation. Such mechanism was demonstrated for many proteins capable of DNA looping, including restriction endonucleases [32,38,39] and transposases [40][41][42][43]. In the alternative scenario, the DNA looping involves (i) binding of a single AfAgo monomer to the rst DNA end (species "4", Fig.…”

“…The measurement of single surface-immobilized molecules with the excitation in the total internal re ection mode (TIR) was performed on the same setup exploiting its alternative functionality as described previously [32]. Brie y, the objective was 100× 1.4 Oil Plan Apo VC (Nikon), the uorescence signal was split by T640lpxr-UF2 dichroic mirror (Chroma) and the different spectral channels were projected on the same EMCCD (DU-897ECS0-UVB, Andor).…”

“…Measurements of surface-immobilized DNA fragments were performed in a ow cell assembled from a six-channel Sticky-Slide VI 0.4 (Ibidi) and a coverslip functionalized with PEG (polyethylene glycol) derivatives as described in detail in [32]. The ow cell was incubated with 5 µg/ml of Neutravidin (Molecular probes) in RB for 2 min, washed with RB, incubated with 5 pM DNA in RB until the density of the surface-immobilized DNA fragments appeared to be appropriate, and washed with RB.…”