Iodouracil-mediated photocrosslinking of DNA to EcoRII restriction endonuclease in catalytic conditions

Abstract

We used a XeCl excimer laser with 50 ns pulses, a frequency of 0.3 Hz and a wavelength of 308 nm in appropriate conditions for the photocrosslinking of EcoRII restriction endonuclease to a 14-mer DNA duplex, containing a 5-iodo-2′-deoxyuridine residue (IdU). IdU replaced the thymidine residue within the EcoRII recognition sequence 5′-CCT/AGG. The binding of EcoRII endonuclease to the IdU-containing DNA duplex was analyzed by gel retardation assay in the presence of Ca²⁺ or Mg²⁺ ions. Photocrosslinking of EcoRII to the IdU-containing DNA duplex occurred in a pre-reactive complex formed in the presence of Ca²⁺ ions. Photocrosslinking yields as a function of time and UV-laser light intensity were studied.