CROSS-LINKING OF PROGESTERONE-RECEPTOR TO DNA USING TUNABLE NANOSECOND, PICOSECOND AND FEMTOSECOND UV LASER-PULSES

UV laser crosslinking is a potentially powerful tool to investigate tr ansient DNA-protein interactions and binding kinetics in intact cells. As the processes underlying UV laser crosslinking are not fully under stood, we have performed a study of the influence of laser pulses with different physical parameters on crosslinking of the progesterone rec eptor to an oligonucleotide containing a hormone-responsive element. W e also studied the influence of the various parameters on the amount o f laser-irradiated DNA that can be correctly primer extended as an ope rational measurement of DNA integrity. A strong influence of pulse int ensity and pulse length on the crosslink yield was found, likely due t o a change in the 'two photon' processes responsible for crosslinking. The highest efficiency of protein crosslinking to DNA was achieved wi th femtosecond pulses and should be sufficient to enable use of this t echnique for in vivo studies.