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Mapping protein-protein interactions between MutL and MutH by cross-linking.

Luis Giron-Monzon·Laura Manelyte·Robert Ahrends·Dieter Kirsch·Bernhard Spengler·Peter Friedhoff
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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.

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Adenosine TriphosphatasesAdenosine TriphosphateAmino Acid SequenceBase Pair MismatchBinding SitesCross-Linking ReagentsCysteineDNA RepairDNA Repair EnzymesDNA-Binding ProteinsDimerizationElectrophoresis, Polyacrylamide GelEndodeoxyribonucleasesEscherichia coliEscherichia coli ProteinsGenetic Complementation TestHistidineLightMass SpectrometryModels, ChemicalModels, MolecularModels, StatisticalMolecular Sequence DataMutL ProteinsMutagenesis, Site-DirectedMutationPlasmidsProtein BindingProtein ConformationProtein Structure, TertiaryTrypsinJournal Article

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Luis Giron-Monzon, Laura Manelyte, Robert Ahrends, Dieter Kirsch, Bernhard Spengler, Peter Friedhoff. 2004-09-14. Mapping protein-protein interactions between MutL and MutH by cross-linking.. https://doi.org/10.1074/jbc.m409307200

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