Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes.
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.
Wong
D L
DL
Department of Chemistry and Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, CA 93106, USA.
Pavlovich
J G
JG
Reich
N O
NO
D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D014498:Uracil; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C048747:5-iodouracil