Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

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 MutH⅐MutL complex and to explain the ATP dependence of this interaction.DNA repair systems are crucial for maintaining a stable genome (1). One of these systems, DNA mismatch repair, is present in most organisms and enhances the fidelity of DNA replication by a factor of up to 1000 by correcting replication errors (2-6). mismatch repair is a complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins. The paradigm of such a process is the MutHLS system from Escherichia coli (7). Because mismatch repair proteins MutS and MutL are evolutionarily conserved among the three kingdoms of life, it is believed that the basic mechanisms of mismatch repair are similar. In the presence of ATP and a mismatch, MutS recruits MutL and together they activate MutH, which in turn cleaves the newly replicated daughter strand at a transiently unmethylated d(GATC) site. The nick marks the erroneous strand for excision, which is started by the action of DNA helicase II, single-stranded DNA-binding protein, and one of several exonucleases (8). DNA polymerase III, single-stranded DNA-binding protein, and DNA ligase carry out repair synthesis.One of the interesting questions in DNA mismatch repair is how the different Mut proteins interact in time and space. In the E. coli system, the assembly of the active MutS⅐MutL complex will activate the strand discrimination endonuclease MutH. Both MutS and MutL are ATPases, and the roles of ATP binding and hydrolysis are just emerging (9 -13). The mismatch activated form of this complex will stimulate the latent endonuclease activity of MutH to cleave the transiently unmethylated error-containing daughter strand, thereby marking this strand for removal. We have previously shown that the proper discrimination of the unmethylated strand from the methylated strand can be mapped to a single amino acid residue in MutH, i.e. Tyr 212 . Biochemical analyses indicated that only MutL, not MutS, interacts direc...

Functional properties of the thermostable mutL from Thermotoga maritima

“…However, in the absence of ADPnP, TmL bound to ssDNA with an apparent KD value of 53.75 ± 5.32 nM and, in the presence of ADPnP, exhibited a higher KD of 25.65 ± 8.47 nM, with a relatively high error-range. These KD values were larger than those form previous E. coli MutL studies (10), but the present results were consistent with previously reported data regarding the ability of bacterial MutL to bind ssDNA in the presence of nucleotides (10,12,13).…”

“…Homodimeric interactions at the N-terminal region of E. coli MutL has been previously shown and single-cysteine E. coli MutL variants found to be crosslinked with the bifunctional linkers bis-maleimidoethane and 1,11-bis-maleimidotetraethyleneglycol, both in the presence and absence of nucleotides (13). A crosslinking assay of the E. coli MutL and its N-terminal domain showed that incubation with ADPnP affects its conformational changes and multimerization (11).…”

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

“…These models were filtered using the experimentally derived distance constraints (this work and Ref. 37). For details of the procedure, see the supplemental information.…”

CYP2C8 Exists as a Dimer in Natural Membranes

“…For cross-linking experiments with BMOE, 200 l of membrane samples adjusted to 1 g of protein/l were mixed with BMOE at a final concentration of 0.4 mM at 37°C or room temperature. At different time points, 20-l aliquots were withdrawn, and the reaction was quenched by addition of DTT to a final concentration of 50 mM and further incubation for 15 min (Giron-Monzon et al, 2004). Samples were analyzed by SDS-PAGE under nonreducing conditions.…”