Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes
| Autor: | Norbert O. Reich, James G. Pavlovich, David L. Wong |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Site-Specific DNA-Methyltransferase (Adenine-Specific)
Protein mass spectrometry Photochemistry Ultraviolet Rays Electrospray ionization EcoRI Biology Protein degradation Mass spectrometry Mass Spectrometry Substrate Specificity chemistry.chemical_compound Genetics Uracil Chromatography High Pressure Liquid Chromatography Binding Sites Base Sequence DNA Molecular biology Thymine Cross-Linking Reagents chemistry Oligodeoxyribonucleotides Covalent bond biology.protein Electrophoresis Polyacrylamide Gel Research Article |
| Zdroj: | Nucleic acids research. 26(2) |
| ISSN: | 0305-1048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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