Order out of disorder: Regulation of endonuclease activity during eukaryotic mismatch repair

The polymerases that copy DNA are very good at their job, pairing adenines with thymines and cytosines with guanines at breakneck speed. Their occasional errors are mostly corrected by proofreading functions built into the polymerase itself and by post-replication mismatch repair (MMR). These keep the frequency of mutation low enough to maintain the health of individuals but high enough to allow for the evolution of populations. MMR is a four-step process: recognition of the mismatched base pair, endonucleolytic cleavage of the strand containing the mis-inserted base, exonucleolytic removal of the base and its neighbors, and strand re-synthesis. These steps rely on a precise choreography of protein-protein and protein-DNA interactions, powered by cycles of ATP binding and hydrolysis in several of the proteins.1 Mismatch recognition is coupled to downstream events by homologs of the Escherichia coli MMR protein, MutL. In E. coli, MutL activates a separate endonuclease but in most other species it is the responsibility of the MutL homologs themselves to cleave the error-containing strand; in eukaryotes this is usually a dimer of Mlh1 and Pms1. The two proteins associate through their C-terminal domains (CTDs), but their N-terminal domains (NTDs) only dimerize during MMR in response to ATP binding. NTD dimerization causes the unstructured linkers that connect the NTD with the CTD in each monomer to wrap around each side of the DNA, forming a ring that holds the dimer in place. Structural rearrangement of the linkers also activates endonuclease activity,2 but the mechanism is unclear. Recent research identified an evolutionarily conserved stretch of amino acids in the linker of the yeast Mlh1 protein that is essential for endonuclease activity in vitro and MMR in vivo.3 The paper by Putnam and Kolodner in this issue of BioEssays4 presents an intriguing hypothesis to explain the role of this motif in licencing endonuclease activity during eukaryotic MMR. The Mlh1 endonuclease active site lies in its CTD. The cleavage reaction is not yet well characterized, but comparisons of the structure of the active site in multiple species suggested to the authors that enzymatic activity is inhibited by a specific cysteine, part of a Phe Glu Arg Cys (FERC) sequence close to the protein's C-terminus. This led them to hypothesize that the linker motif activates the enzyme during MMR by displacing the inhibitory cysteine. They justify their hypothesis by using structural and evolutionary data to associate the linker motif with the active site and with the FERC sequence. Published data from in vitro cross-linking studies4 had shown that the linker motif interacts with the CTDs of the Mlh1-Pms1 dimer. By searching for sequences in the two proteins that had co-evolved with the linker motif, the authors placed the interaction site in a region of the Mlh1 CTD close to the endonuclease active site. In parallel, they established a relationship between the linker motif and FERC by showing that these two have also co-evolved; with very few exceptions, all eukaryotic Mlh1 proteins for which sequence data is available have both the FERC sequence and the linker motif, while other MutL homologs have neither. The authors propose experiments to determine whether the structural links that they have made between the inhibitory cysteine and the linker motif have relevance for regulation of endonuclease activity and MMR. MutL homologs are arguably the most important components of MMR, transducing signals that originate with recognition of the mismatch by the MutS homologs to the nucleases, helicases and polymerase that operate on the DNA to restore its correct sequence. They also remain the most enigmatic, in part because of the conformational contortions that they undergo during the repair process. So, a testable hypothesis for how reorganization of the intrinsically disordered region (IDR) that connects ATP binding and hydrolysis in the NTD to regulation of endonuclease activity in the CTD is valuable for understanding how the many steps in MMR occur at the right time and in the right order. Such understanding would also contribute to our knowledge of how IDRs in many eukaryotic proteins function in signal transduction.5 This article comments on the hypothesis paper by Christopher Putnam and Richard Kolodner, https://doi.org/10.1002/bies.202300031. The author declares no conflicts of interest.

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