title: Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions.
abstract: To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433- 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.
authors.affiliation: Department Lehrach, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany. birgit.kersten-berlin@gmx.de
authors.forename: Birgit
authors.initials: B
authors.lastname: Kersten
authors.forename: Alexandra
authors.initials: A
authors.lastname: Possling
authors.forename: Franca
authors.initials: F
authors.lastname: Blaesing
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Mirgorodskaya
authors.forename: Johan
authors.initials: J
authors.lastname: Gobom
authors.forename: Harald
authors.initials: H
authors.lastname: Seitz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D040081:Protein Array Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D017931:DNA Primers