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

We used a XeCl excimer laser with 50 ns pulses, a frequency of 0.3 Hz and a wavelength of 308 mn 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.We thank Professor Alexander Yu. Borisov for valuable discussions and Robin Rycroft for assistance in preparation of the manuscript. We are grateful to Professor Ashok Bhagwat for providing us with the pR224 plasmid containing the ecoRII gene. This study was supported by the Russian Foundation of Fundamental Investigation (project no. 01–04–48637).Peer reviewe