Russmann C, Truss M, Fix A, Naumer C, Herrmann T, Schmitt J, Stollhof J, Beigang R, Beato M
Institut für Molekularbiologie und Tumorforschung, Emil-Mannkopff-Strasse 2, D-35033 Marburg, Germany.
Nucleic Acids Res. 1997 Jun 15;25(12):2478-84. doi: 10.1093/nar/25.12.2478.
UV laser crosslinking is a potentially powerful tool to investigate transient DNA-protein interactions and binding kinetics in intact cells. As the processes underlying UV laser crosslinking are not fully understood, we have performed a study of the influence of laser pulses with different physical parameters on crosslinking of the progesterone receptor to an oligonucleotide containing a hormone-responsive element. We also studied the influence of the various parameters on the amount of laser-irradiated DNA that can be correctly primer extended as an operational measurement of DNA integrity. A strong influence of pulse intensity and pulse length on the crosslink yield was found, likely due to a change in the 'two photon' processes responsible for crosslinking. The highest efficiency of protein crosslinking to DNA was achieved with femtosecond pulses and should be sufficient to enable use of this technique for in vivo studies.
紫外激光交联是研究完整细胞中瞬时DNA-蛋白质相互作用和结合动力学的一种潜在有力工具。由于紫外激光交联的潜在机制尚未完全了解，我们开展了一项研究，考察具有不同物理参数的激光脉冲对孕酮受体与含有激素反应元件的寡核苷酸交联的影响。我们还研究了各种参数对可正确进行引物延伸的激光照射DNA量的影响，以此作为DNA完整性的一项操作指标。结果发现脉冲强度和脉冲长度对交联产率有很大影响，这可能是由于负责交联的“双光子”过程发生了变化。飞秒脉冲实现了蛋白质与DNA的最高交联效率，这应该足以使该技术用于体内研究。
