PMID:15371440

Citation	Giron-Monzon, L, Manelyte, L, Ahrends, R, Kirsch, D, Spengler, B and Friedhoff, P (2004) Mapping protein-protein interactions between MutL and MutH by cross-linking. J. Biol. Chem. 279:49338-45
Abstract	Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.
Links	PubMed Online version:10.1074/jbc.M409307200
Keywords	Adenosine Triphosphatases/chemistry; Adenosine Triphosphate/chemistry; Amino Acid Sequence; Base Pair Mismatch; Binding Sites; Cross-Linking Reagents/pharmacology; Cysteine/chemistry; DNA Repair; DNA Repair Enzymes/chemistry; DNA-Binding Proteins/chemistry; Dimerization; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases/chemistry; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Genetic Complementation Test; Histidine/chemistry; Light; Mass Spectrometry; Models, Chemical; Models, Molecular; Models, Statistical; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Plasmids/metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Trypsin/pharmacology
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