RESUMO
Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential 16O/18O-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry.
Assuntos
Proteínas Fúngicas/química , Espectrometria de Massas/métodos , Nucleoproteínas/análise , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Tripsina/química , Análise de Dados , Marcação por Isótopo , Nucleoproteínas/química , Oxigênio/química , Isótopos de Oxigênio/química , Software , Fluxo de TrabalhoRESUMO
UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides, and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterization of the specific sites of cross-linking is extremely challenging. As a tool for the optimization of sample preparation, ionization, fragmentation, and detection by mass spectrometry, novel synthetic DNA-peptide heteroconjugates were generated to act as mimics of UV cross-linked heteroconjugates. Click chemistry was employed to cross-link peptides to DNA oligonucleotides. These heteroconjugates were fully characterized by high resolution FTICR mass spectrometry and by collision-induced dissociation (CID) following nuclease P1 digestion of the DNA moiety to a single nucleotide monophosphate. This allowed the exact site of the cross-linking within the peptide to be unambiguously assigned. These synthetic DNA-peptide heteroconjugates have the potential to be of use for a variety of applications that involve DNA-peptide heteroconjugates.
Assuntos
Química Click , DNA/química , Espectrometria de Massas , Peptídeos/química , Catálise , Cobre/química , Estrutura MolecularRESUMO
Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.