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 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.
Babkina
Olga V
OV
Department of Chemistry and Belozersky Institute of Physical and Chemical Biology, Moscow State University, Moscow 119992, Russia.
Chutko
Catherine A
CA
Shashkov
Alexander A
AA
Dzhidzhoev
Murat S
MS
Eritja
Ramon I
RI
Gromova
Elizaveta S
ES
D001483:Base Sequence; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism / Q000528:radiation effects*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; C100034:iodouracil; D014498:Uracil; D004247:DNA; D004262:DNA Restriction Enzymes; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific