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

Abstract

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.

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

“…Interestingly, heterogeneity of NgoMIV‐induced DNA loop stabilities observed in the current work was not encountered in our previous study on another type IIF REase—Ecl18kI . Unlike homotetrameric NgoMIV, Ecl18kI is a homodimer in the DNA‐free form, and Ecl18kI‐mediated DNA looping occurs through two Ecl18kI homodimers bound to specific recognition sequences associating to form a tetramer bridging the two cognate sites .…”

“…We used a custom‐built single‐molecule fluorescence microscopy setup described in detail previously . In brief, the setup is based on a commercial inverted Nikon Eclipse Ti‐U microscope, equipped with 100× 1.4 Oil Plan Apo VC objective, EMCCD detector (DU‐897E‐CS0‐UVB, Andor) and 25 mW 532 and 635 nm diode‐pumped solid state and diode lasers, respectively (Crystalaser).…”

“…We used a custom-built single-molecule fluorescence microscopy setup described in detail previously. [12,13] In brief, the setup is based on a commercial inverted Nikon Eclipse Ti-U microscope, equipped with…”

“…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.…”

“…[9][10][11] In our own work, we have employed SM fluorescence microscopy to explore the DNA looping by a dimeric REase Ecl18kI. [12,13] 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, [14,15] capable of simultaneous binding of two 5 0 -GCCGGC-3 0 target sites via two identical DNA-binding surfaces.…”