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

Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2004; 279(47):49338-45. DOI: 10.1074/jbc.M409307200
Source: PubMed

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.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Plasmid partition systems require site-specific DNA binding proteins to recognize the plasmid partition site, or centromere. When bound to the centromere, these proteins, typically called ParB, interact with the ParA ATPases, which in turn promote the proper positioning of plasmids prior to cell division. P1 ParB is a typical member of a major class of ParB-like proteins that are dimeric helix-turn-helix DNA binding proteins. The N-terminus of ParB contains the region that interacts with ParA and with itself, but it has been difficult to study because this region of the protein is flexible in solution. Here we describe the use of cysteine-scanning mutagenesis and thiol modification of the N-terminus of ParB to create tools to probe the interactions of ParB with itself, with ParA and with DNA. We introduce twelve single-cysteine substitutions across the N-terminus of ParB and show that most do not compromise the function of ParB and that none completely inactivate the protein in vivo. We test three of these ParB variants in vitro and show that they do not alter ParB function, measured by its ability to stimulate ParA ATPase activity and its site-specific DNA binding activity. We discuss that this approach will be generally applicable to the ParB-like proteins in this class of partition systems because of their natural low content of cysteines, and because our evidence suggests that many residues in the N-terminus are amenable to substitution by cysteine.
    Plasmid 02/2013; DOI:10.1016/j.plasmid.2013.02.002 · 1.76 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This review is focused on the general aspects of the DNA mismatch repair (MMR) process. The key proteins of the DNA mismatch repair system are MutS and MutL. To date, their main structural and functional characteristics have been thoroughly studied. However, different opinions exist about the initial stages of the mismatch repair process with the participation of these proteins. This review aims to summarize the data on the relationship between the two MutS functions, ATPase and DNA-binding, and to systematize various models of coordination between the mismatch site and the strand discrimination site in DNA. To test these models, novel techniques for the trapping of short-living complexes that appear at different MMR stages are to be developed.
    01/2013; 5(3):17-34.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mutator strains spontaneously arise in bacterial populations under stress in an attempt to increase evolutionary adaptation. Inactivation of the ubiquitous DNA mismatch repair pathway, whose normal function is to correct replication errors and hence increase replication fidelity, is often the cause of the mutator phenotype. One of the essential genes in this pathway, mutL, includes a short tandem repeat that is prone to polymerase slippage during replication. While extensive work has established that this repetitive sequence is a genuine genetic switch, the mechanism of MutL inactivation remains unclear. This short tandem repeat is translated into a LALALA motif that resides near the ATPase active site of MutL. Therefore, changes in the length of this motif are presumed to alter the ATPase activity of MutL. We have engineered variants of Escherichia coli MutL with shorter/longer LALALA motifs and characterized their ATPase and DNA binding functions. We have found that the deletion or insertion of a single LA repeat did not compromise the structural integrity of the protein, nor did it affect MutS- or DNA-binding activity. However, it severely compromised ATP binding and, consequently, engagement of the N-terminal domains; both essential activities for proper DNA mismatch repair. These results are discussed in the context of the structure of MutL.
    DNA repair 08/2013; DOI:10.1016/j.dnarep.2013.07.003 · 3.36 Impact Factor