Identification of Tyrosine 204 as the Photo-Cross-Linking Site in the DNA−EcoRI DNA Methyltransferase Complex by Electrospray Ionization Mass Spectrometry

• Published in: Biochemistry (Easton) 2000-12, Vol.39 (50), p.15410-15417
• Main Authors: Wong, David L, Reich, Norbert O
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Cross-Linking Reagents; DNA - chemistry; DNA - metabolism; DNA methyltransferase EcoRI; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; Escherichia coli; Protein Binding; Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001164v

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein−DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA−protein complex at 313 nm results in a >60% cross-linking yield. SDS−polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide−DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide−nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of “DNA binding” motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA−protein complexes.