This method can help decipher the steps in DNA strand exchange and reveals the molecule roles of different proteins involved in combination during repair. With this technique we can monitor DNA strand exchange in real time without any disruption to the reaction and use the operating theater to determine the kinetics of each reaction step. With some minor adjustments, this technique can be applied to study the activities of purified recombination proteins derived from other species, such as mammals and plants.
Demonstrating the procedure will be Kentaro Ito, a Post Doc from my laboratory. To begin, prepare the reaction buffer A according to the text protocol. Next, add 36-nanomole 16FA-oligonucleotide to the buffer for the DNA pairing assay.
Incubate the mixture at 37 degrees Celsius for five minutes. Add RAD-51 protein at a final concentration of 1.5 micro moles to the mixture and incubate it at 37 degrees celsius for five minutes. Add SWI5-SFR1 protein at a final concentration of 0.15 micro moles to the mixture, and incubate it at 37 degrees Celsius for an additional five minutes.
Transfer 1.5 milliliters of the mixture in a 1.0 by 1.0 cm quartz cuvette containing a magnetic stirrer. Insert the cuvette into a spectrofluorometer and proceed to adjusting the temperature controller and the magnetic stirrer. Record the change in FAM fluorescence emission at 525 nanometers upon excitation at 493 nanometers at one second intervals for 100 seconds.
Finally, with a syringe, inject ROX labeled double-stranded DNA at a final concentration of 36 nano moles into the cuvette. Record the change in fluorescence emission at one second intervals for 30 minutes. To compare the fluorescence spectra between the substrates and the final products, insert each cuvette containing the 16 FA-oligonucleotides with or without RAD-51 into a spectrofluorometer and incubate at 37 degrees Celsius for five minutes.
Then, record the FAM fluorescence emission at 500 to 600 nano meters upon excitation at 493 nanometers. To test the effect of RAD-51 on the fluorescence spectra, add RAD-51 at a final concentration of 1.5 micro moles. And incubate the mixture at 37 degrees Celsius for five minutes.
Finally, record the fluorescence spectra from 500 to 600 nanometers upon excitation at 493 nanometers. The maximum FRET efficiency is obtained by measuring the maximum reduction in the fluorescence intensity when all single-stranded DNA substrate is converted into double-stranded DNA in the pairing assay. Or by measuring the maximum increase in the intensity when all double-stranded DNA substrate is converted into single-stranded DNA in the displacement assay.
In both assays, addition of RAD-51 protein did not affect fluorescence emission of FAM, or its quenching efficiency by rocks. Effects of the spontaneous reactions between substrate DNAs and the subsequent photo bleaching are small. As shown by the negligible changes in the emission of FAM without RAD-51, compared to the substantial changes seen with RAD-51.
Adding the SWI5-SFR1 complex strongly stimulates the pairing activity of RAD-51. The pairing reaction is simulated using a three step model, consisting of formation of the first three-strand intermediate, transition into the second intermediate, and release of the single-stranded DNA and formation of the hetero duplex. A comparison of the three step model with a two step model indicates that the three step model is a better fit for simulating DNA pairing, without or with SWI5-SFR1 complex.
The calculated equilibrium constant of each reaction step, with or without SWI5-SFR1, shows that the SWI5-SFR1 complex does not stimulate the formation of the first three-strand intermediate, but strongly stimulates the transition between the two three-strand intermediates, and the release of SS DNA. It is essential to verify that each batch of purified protein is free of nuclease and helicase contamination.
