To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.
The genetic code of DNA is written using four letters: “A”, “C”, “T”, and “G”. Molecules of DNA form a double helix in which the letters in the two opposing strands pair up in a specific manner—“A” pairs with “T”, and “C” pairs with “G”. A cell must replicate its DNA before it divides, and sometimes the wrong DNA letter can get added into the new DNA strand. If left uncorrected, these mistakes accumulate over time and can eventually harm the cell. As a result, cells have evolved several ways to identify these mistakes and correct them, including one known as “mismatch repair”.
Mismatch repair occurs via several stages. The process starts when a protein called MutS comes across a site in the DNA where the letters are mismatched (for example, where an “A” is paired with a “C”, instead of a “T”). MutS can recognize such a mismatch, bind it, and then bind to another molecule called ATP. MutS then changes shape and encircles the DNA like a clamp that can slide along the DNA. Only when it forms this “sliding clamp” state can MutS recruit another protein called MutL. This activity in turn triggers a series of further events that ultimately correct the mismatch. However, it remains poorly understood how MutS forms a clamp around DNA and how and why this state recruits MutL in order to start the repair.
To visualize this short-lived intermediate, Groothuizen et al. trapped the relevant complex in the presence of DNA containing a mismatch and then used a technique called X-ray crystallography to determine the three-dimensional structure of MutS bound to MutL. The structure reveals that two copies of MutS tilt across each other and open up a channel, which is large enough to accommodate the DNA. In this manner, MutS is able to form a loose ring around the DNA. The changes in the structure and the movement of the DNA to the new channel were confirmed using another technique, commonly referred to as FRET.
Groothuizen et al. observed that the movements in the MutS protein also serve to make the interfaces available that can recognize MutL. If these interfaces were disturbed, MutS and MutL were unable to associate with each other, which resulted in a failure to trigger mismatch repair. Further analysis revealed that that MutL binds to DNA only after MutS has recognised the mismatch and formed a clamp around it. This is the first time that the MutS clamp and the MutS/MutL complex have been visualized, and further work is now needed to understand how MutL triggers other events that ultimately repair the mismatched DNA.