Two wavelength high intensity irradiation for effective crosslinking of DNA to protein

by Guler, Emine.

Protein-DNA crosslinking is an important method to study protein- DNA interactions. Crosslinking by short pulsed UV lasers is a potentially powerful tool that results in efficient crosslinking, apparently by a two photon process. However, the major problem in using UV laser crosslinking is that the conditions which lead to high crosslinking efficiency also result in high DNA damage. Previously, it has been shown that a combination of femtosecond laser pulses at two different wavelengths, in the UV (266 nm) and the visible range (400 nm), increases the effective crosslinking yield (i.e. higher crosslinking yields with reduced DNA damage). This new strategy has the advantage that the intensity of the UV pulse for the first excitation step can be kept low, leading to lower UV-induced DNA damage and the second pulse at a visible wavelength can provide enough energy for the UV excited bases to cross their ionization threshold without damaging the DNA . The objective of this thesis project was to develop a novel UV laser cross-linking technique that would permit higher effective crosslinking yields with the commonly used pulses in the nanosecond (ns) 7 range. To serve this purpose we tried to extend the two-wavelength femto second laser irradiation approach to longer duration pulses. We chose MBP-PIF3 protein and its target G-box DNA motif as a model system. Before ultraviolet irradiation of the protein-DNA complexes in vitro, the specific binding interaction of purified MBP-PIF3 protein with the G-box DNA motif was studied by Electrophoretic Mobility Shift Assay (EMSA). We irradiated the PIF3/DNA complexes with different laser systems (i.e. Nd:YAG and Dye lasers) and their combinations. We were expecting to see that the combination of UV laser pulses (260nm) with longer wavelength dye laser pulses (480nm) will produce higher effective crosslink yields relative to the yield from the UV pulses alone. However we could not detect any crosslinked MBP-PIF3/DNA bands by denaturing SDS-PAGE after irradiation of protein-DNA complex with UV laser pulses (266 nm, 5 ns, and ~5 mJ/pulse) alone or with UV (266 nm, 5 ns, and ~5 mJ/pulse) and blue laser pulses (480 nm, 800ns, and 60 mJ/pulse) together. 8 It may be necessary in the future to experimentally determine the optimum range of photon flux for the PIF3/DNA complex to obtain maximum amount of crosslinking. Alternatively, this new approach could be tested on a different DNA-binding protein that has a greater propensity to undergo UV-promoted DNA crosslinking. 9