Covalently Trapping of MutS on DNA

The highly conserved DNA mismatch repair (MMR) system is responsible for recognition and removal of DNA damages that arise during DNA replication (1,2). A failure of MMR results in a higher mutation rate finally leading to cancer in humans (3). The principal mismatch repair protein is MutS that scans the DNA for mismatches (4). Bacterial MutS are homodimeric DNA binding proteins with an ABC ATPase. Upon mismatch binding by MutS the DNA is kinked by 60° at the mismatch thereby forming the mismatch recognition complex (5). Mismatch binding triggers an ADP/ATP exchange and prevents ATP hydrolysis finally leading to the formation of a mobile sliding clamp, the activated form of MutS that initiates the subsequent repair reactions. With exception for the mismatch recognition complex no structural information regarding the mobile scanning or activated MutS clamps are available. To overcome the shortcomings of a mobile DNA binding protein and to allow analysis of the MutS intermediates in the MMR pathway we used site directed chemical crosslinking in order to trap this highly dynamic complex for further functional and structural studies. We were able to trap MutS on DNA in a mismatch- and nucleotide-dependent manner as well as to purify the MutS-x-DNA complex. Functional analysis revealed that MutS is still active after crosslinking to DNA demonstrating that the trapped MutS-x-DNA complex is an optimal starting point to answer outstanding questions in MMR initiation.