Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking
| Autor: | Bernhard Spengler, Peter Friedhoff, Laura Manelyte, Robert Ahrends, Dieter Kirsch, Luis Giron-Monzon |
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| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular DNA Repair Light Base Pair Mismatch Protein Conformation Molecular Sequence Data Biochemistry Mass Spectrometry Protein–protein interaction Endonuclease Adenosine Triphosphate MutS-1 Escherichia coli Histidine Trypsin Amino Acid Sequence Cysteine Molecular Biology Adenosine Triphosphatases Binding Sites Endodeoxyribonucleases Models Statistical biology Escherichia coli Proteins Genetic Complementation Test Mutagenesis Escherichia coli DNA Cell Biology Protein Structure Tertiary DNA-Binding Proteins Cross-Linking Reagents DNA Repair Enzymes MutL Proteins Models Chemical Mutation Mutagenesis Site-Directed biology.protein Molecular mechanism Electrophoresis Polyacrylamide Gel DNA mismatch repair Dimerization Plasmids Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 279:49338-49345 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m409307200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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