Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Abstract

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems. Video Link The video component of this article can be found at https://www.jove.com/video/60045/ 10. In TIRF, a time-series of a few hundreds of molecules immobilized on the surface is recorded by a position-sensitive two-dimensional charge coupled detector (CCD). The CCD amplifies the fluorescence signal either by intensified phosphor screen and microchannel plate or on-chip multiplication of photoelectrons (EMCCD). The temporal resolution is dependent on the readout speed and quantum efficiency of the CCD and usually on the order of few tens of milliseconds 6. HJ is a central intermediate in DNA repair and recombination 11,12,13,14. HJ has two continuous and two crossing strands that connect between the continuous strands without intersecting each other. HJ exists in solution as X-stacked conformers, which undergo continuous isomerization by the continuous strands becoming crossing and the crossing strands becoming continuous in the other conformer 15. Isomer preference of the HJ is dependent on the core sequence and ionic environment and has been extensively studied by FRET 16,17,18,19. GEN1 20 is a monomeric protein in solution 21 and requires dimerization to cleave the HJ, thus allowing proper separation of the recombined strands 22,23. The stacking conformer preference of the HJ influences the outcome of genetic recombination by setting the orientation of the

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

“…Glass coverslips were functionalized by 1:100 molar ratio of biotinylated PEG to mPEG 91 . The coverslips were used for building a flow cell with one inlet and one outlet tubbing, as previously described 13,14,36,91 .…”

“…Glass coverslips were functionalized by 1:100 molar ratio of biotinylated PEG to mPEG 91 . The coverslips were used for building a flow cell with one inlet and one outlet tubbing, as previously described 13,14,36,91 . Before each experiment, 0.2 mg/mL NeutrAvidin were injected into the flow cells and incubated for 10 min.…”

“…TIRF-based FRET imaging was performed using a custom-built setup described previously 91,94 . Movies were acquired using xCellence rt (Olympus) under continuous excitation (CW) with a green laser, at a temporal resolution of 50, 100, or 160 ms, as indicated for each experiment.…”

“…Movies were acquired using xCellence rt (Olympus) under continuous excitation (CW) with a green laser, at a temporal resolution of 50, 100, or 160 ms, as indicated for each experiment. A transformation matrix file was generated by imagining fluorescent beads to aid particle linking between the green and red emission channels, as previously described 91 . All movies were analyzed using the TwoTone software integrated into MATLAB (MathWorks) as previously described 13,14,36,91,95 .…”

Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

“…Depending on the experimental design and the type of microscopy employed, the duration of observation for each single molecule can range anywhere from milliseconds to minutes [ 43 ], with the maximum temporal resolution ultimately limited by the rate at which photons are detected. For molecular processes that occur on a time-scale much slower than the photon count rate, the conformational dynamics can readily be extracted from the anticorrelated fluctuations in the donor and acceptor fluorescence time traces [ 44 , 45 ]. Furthermore, the relative occupancy of these conformational subpopulations directly reports on the standard state free energy differences between them, providing additional biochemical insights into the process of interest.…”