Journalartikel

Jahr der Veröffentlichung: 2006

Seiten: 3169-3180

Zeitschrift: Nucleic Acids Research

Bandnummer: 34

Heftnummer: 10

ISSN: 0305-1048

Open Access Status: Gold

DOI Link: https://doi.org/10.1093/nar/gkl407

Verlag: Oxford University Press

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 photocrossfinking (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.

Harvard-Zitierstil: Ahrends, R., Kosinski, J., Kirsch, D., Manelyte, L., Giron-Monzon, L., Hummerich, L., et al. (2006) Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry, Nucleic Acids Research, 34(10), pp. 3169-3180. https://doi.org/10.1093/nar/gkl407

APA-Zitierstil: Ahrends, R., Kosinski, J., Kirsch, D., Manelyte, L., Giron-Monzon, L., Hummerich, L., Schulz, O., Spengler, B., & Friedhoff, P. (2006). Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry. Nucleic Acids Research. 34(10), 3169-3180. https://doi.org/10.1093/nar/gkl407