Abstract
To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Amino Acid Sequence
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Benzophenones / chemistry
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Binding Sites
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Chromatography, Affinity
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Cross-Linking Reagents
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Cysteine / genetics
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DNA Repair Enzymes / chemistry*
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DNA Repair Enzymes / metabolism
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / metabolism
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Dimerization
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Endodeoxyribonucleases / chemistry*
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Endodeoxyribonucleases / metabolism
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Evolution, Molecular
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Light
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Maleimides / chemistry
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MutL Proteins
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Mutagenesis, Site-Directed
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Peptide Hydrolases
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Peptides / chemistry
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Peptides / isolation & purification
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Protein Structure, Tertiary
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Sequence Alignment
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Streptavidin / chemistry
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Sulfhydryl Compounds / chemistry
Substances
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Benzophenones
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Cross-Linking Reagents
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DNA-Binding Proteins
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Escherichia coli Proteins
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Maleimides
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MutL protein, E coli
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Peptides
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Sulfhydryl Compounds
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Streptavidin
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benzophenone-4-maleimide
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Endodeoxyribonucleases
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methyl-directed mismatch repair protein, E coli
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Peptide Hydrolases
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Adenosine Triphosphatases
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MutL Proteins
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DNA Repair Enzymes
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Cysteine