LIG_MLH1_MIPbox_1

Organisms have developed robust DNA repair mechanisms to prevent the deleterious consequences of different types of DNA damage. The base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways cooperate in the repair of oxidative DNA damage through protein-protein interactions (Gellon,2002), while the MMR pathway is also responsible for correcting base-base mismatches and insertion/deletion loops arising during DNA replication (Dherin,2009). MutL and MutS are essential factors of MMR and meiotic recombination. MutLα is a heterodimer composed of Mlh1 and Pms1 in yeast and of MLH1 and PMS2 in humans. This dimer is believed to act as a scaffold that links the proteins that recognize the mismatches in the DNA with the proteins that carry out the subsequent steps of MMR. Mlh1 interacts through its C-terminal domain (CTD; PF16413, Mlh1_C Domain) with MutL homologs and proteins involved in DNA repair and replication. One can distinguish two different groups among the partners of the Mlh1 CTD, which employ two different, non-overlapping binding sites for Mlh1 interaction: Pms1, Mlh2, and Mlh3, which are required for specialized MMR functions, interact with site 1 while Exo1, Sgs1, and Ntg2, which are not strictly required for MMR interact with site 2 (Gellon,2002). The MIP-box is a widely conserved short sequence motif that is specific for the partners binding site 2. The motif is composed of a conserved serine residue followed by a preferentially positively charged residue and two aromatic residues, with the second being a phenylalanine. The interactions of MIP-box motifs in different replication and repair proteins with Mlh1 CTD have been confirmed by diverse methods, including two-hybrid studies, pull-down assays, alanine-scanning (Gellon,2002), ITC measurements (Dherin,2009) and the structures of two complexes are also available (Gueneau,2013). X-ray crystal structures of Mlh1 bound to MIP motifs from Exo1 (4FMO) and Ntg2 (4FMN) show that the MIP peptides adopt an ST turn conformation in which the side chain of the serine forms a hydrogen bond with the main chain amine group of the first aromatic residue (Gueneau,2013). Residues in the MIP motif important for Mlh1 binding include the serine and the two aromatic residues. These latter residues bind in hydrophobic pockets (Gellon,2002, Dherin,2009, Gueneau,2013).
Due to the presence of two different sites, Mlh1 can form heterotrimers by binding a MutL homolog and an S2-interacting protein at the same time, for example, the Mlh1-Pms1-Exo1 heterotrimer (Tran,2001). Binding multiple partners at sites S1 and S2 may result in the formation of a variety of binary and ternary complexes, whose biological impact must be diverse. Therefore Mlh1 acts as a “molecular matchmaker,” a major coordinator, and regulator of the MMR proteins and coupling this pathway to replication machinery (Gellon,2002).
In the large network of interacting proteins responsible for genome maintenance, PCNA, Rev1, Mlh1, and pol δ are hubs responsible for the perception and integration of different signals and for the orchestration of adequate responses. Many proteins interact with these hubs via short sequence motifs such as PIP box motifs, RIR motifs, MIP-box motifs, and F1 motifs, respectively. The traditional paradigm is that these motifs are distinct entities, each specific for binding a single target protein. However, the motifs have consensus sequences that involve two adjacent aromatic residues that facilitate the interactions with target proteins by binding within hydrophobic pockets. Interestingly, several studies have challenged this paradigm by showing that some of these simple double-aromatic residue motifs are specific for more than one target protein (Boehm,2016). For example, the RIR motif of human polymerase η interacts with both Rev1 and pol δ (Baldeck,2015). Similarly, the PIP motif of yeast pol η interacts with both PCNA and Rev1 (Boehm,2016). Furthermore, the PCNA-binding motif on the N-terminus of yeast MutSβ subunit Msh3 was demonstrated to be critical for MutSβ – MutLα binding and thus it presumably also acts as a MIP motif binding to the Mlh1 subunit of MutLα (Mlh1-Pms1 heterodimer) (Iyer,2010). The ability of these peptide motifs to bind more than one target proteins may necessitate to re-think their strict classifications and to introduce a broader class of PIP-like motifs (Boehm,2016), perhaps with molecular switching properties.