Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography.
The RuvABC resolvasome of Escherichia coli typifies nucleoprotein complexes involved in genetic transactions. This molecular assembly catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) to rapidly identify the binding of RuvA to an immobilised synthetic Holliday junction; unambiguous identification was verified using tryptic digest of the bound protein. In conjunction with a novel fluorescent-based technique incorporating ion pair reverse phase liquid chromatography, a "footprint" of the RuvA:Holliday complex was obtained. These two complementary techniques offer a generic approach to the analysis of nucleoprotein complexes.
Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Mark J
MJ
Dickman
Svetlana E
SE
Sedelnikova
John B
JB
Rafferty
David P
DP
Hornby
D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000032:analysis / Q000378:metabolism; D045566:DNA, Cruciform / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011506:Proteins; D017665:Hydroxyl Radical; D004247:DNA; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases