A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry

Abstract

Photochemical crosslinking is a method for studying the molecular details of protein-nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe3+-IMAC (immobilized metal affinity chromatography) purification of peptide-oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein-DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide-oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe3+-IMAC. A combination of MS analysis of the peptide-nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys209 of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys209 is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.

Figure 1

Outline of our protocol for the identification of the crosslinked amino acid.

Figure 2

Analytical photocrosslinking of MboI and oligodeoxynucleotide: SDS–PAGE analysis of the time course of the photocrosslinking reaction with MboI and a 20 bp oligodeoxynucleotide (US20/LS20) monosubstituted with 5-IdU. The MboI–oligodeoxynucleotide complex was formed by incubating 10 μM MboI dimer with 10 μM ³²P-labeled oligodeoxynucleotide containing the 5-IdU residue in the position of the adenine in the top strand of the recognition sequence for 15 min at ambient temperature in a volume of 100 μl. The mixture was irradiated and 5 μl aliquots were withdrawn after the indicated time periods. The silver-stained gel of the samples analyzed by 15% SDS–PAGE is shown. S represents the molecular mass standard.

Figure 3

SDS–PAGE analysis of the preparative MboI–DNA crosslink experiment. (A) An aliquot of 100 pmol of the MboI–DNA complex were analyzed before and after irradiation for 45 min at 325 nm (lanes a and b), a second irradiated sample was loaded after 45 min. Only 2.5 μl of the 100 μl reaction volume was used for the analysis. The gel was stained by Coomassie Brilliant Blue. (B) The autoradiogram of the gel is shown.

Figure 4

Urea gel electrophoresis. Aliquots of the MboI–DNA adducts were analyzed before (lane 1) and after enzymatic degradation reactions with trypsin (lane 2), trypsin and chymotrypsin (lane 3) and with proteinase K (lane 4) on a 15% polyacrylamide gel containing 0.5× TBE and 2 M urea. Bands corresponding to the undigested MboI–DNA adduct (CL-MboI), peptide–DNA adducts and free oligodeoxynucleotide are indicated.

Figure 5

MALDI-TOF mass spectra of the dU-containing peptides obtained after elution of the proteolytic peptide–DNA heteroconjugates from the Fe³⁺-IMAC column and after HF treatment. Signals correspond to protonated pseudomolecular ions [M + H]⁺ of the tryptic peptide dU-TYVIETNYYNSGGSKLNEVAR at m/z 2604.42 (A), the tryptic/chymotryptic peptide dU-NSGGSKLNEVAR at m/z 1457.52 (B) and the peptide dU-GGSKLN at m/z 801.17 resulting from digestion with proteinase K (C).

Figure 6

Identification of the crosslinked amino acid residue in MboI by MALDI-TOF-MS/MS of the tryptic/chymotryptic dU-peptide (m/z 1457.52). Sequence-specific peptide ions are labeled according to Roepstorff and Fohlman (46) and Biemann (47). Deduced amino acid sequences are included.