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

Ines Winkler,Andreas D. Marx,Damien Lariviere,Michele Cristovao,Ute Curth,Annet Reumer,Roger J. Heinze,Peter Friedhoff,Titia K. Sixma

doi:10.1074/jbc.m110.187641

Abstract

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.

Highlights

Bacterial MutS exists in a dimer-tetramer equilibrium, but the dimeric form of the protein is sufficient for DNA mismatch repair [8, 9]
In E. coli, the mismatched base is stacked onto Phe-36 and hydrogen-bonded to Glu-38, and stacking of Phe-36 between the bases is important for mismatch recognition and prevents MutS from sliding on the DNA [20]
Cys-93 in Mismatch-binding Domain of MutS Cross-links to Cys-131 in ATPase Domain of MutL—In a previous report, we demonstrated that the single cysteine MutL variant (MutL131C) modified with benzophenone at Cys-131 in the ATPase domain could be photo-cross-linked to MutS [43]

Summary

Introduction

Bacterial MutS exists in a dimer-tetramer equilibrium, but the dimeric form of the protein is sufficient for DNA mismatch repair [8, 9]. ATP and Mg2ϩ binding was shown to be crucial for the formation of a sliding clamp and for the formation of a dynamic ternary complex comprising DNA-MutS-MutL [27, 28]. This ternary complex coordinates all subsequent steps in DNA repair (e.g. strand discrimination and DNA unwinding) and is involved in signaling the DNA mismatch/damage to other cellular responses [29]. Mutational analyses and hydrogen/deuterium exchange mass spectrometry analyses suggested that the connector domain of MutS contains residues critical for the interaction with MutL [32]

Results

Discussion

Conclusion