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Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

MPG-Autoren

Kersten,  Birgit
Max Planck Society;

Possling,  Alexandra
Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50102

Blaesing,  Franka
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

Mirgorodskaya,  Ekaterina
Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50168

Gobom,  Johan
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50550

Seitz,  Harald
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Zitation

Kersten, B., Possling, A., Blaesing, F., Mirgorodskaya, E., Gobom, J., & Seitz, H. (2004). Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions. Analytical Biochemistry, 331(2), 303-313.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-87EF-0
Zusammenfassung
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.