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Nucleic Acids Research, Vol 26, Issue 2 645-649, Copyright © 1998 by Oxford University Press

ARTICLES

Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes

DL Wong, JG Pavlovich and NO Reich
Department of Chemistry and Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, CA 93106, USA.

We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. Eco RI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5- iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5- iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies.
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