Fig 2: Rad5 interacts with Mlh1 through a MIP box motif but does not interact with Msh2 through a SHIP box motif. (A) The MIP box and SHIP box motif matching scores for every 7mer peptide in S. cerevisiae Rad5 is plotted against the predicted disorder score, showing that the predicted MIP and SHIP boxes have the best peptide scores and are predicted to be disordered by IUPRED (Dosztanyi et al., 2005). (B) Sequence logos of the first 47 amino acids of S. cerevisiae Rad5 generated by Seq2Logo (Thomsen and Nielsen, 2012) were calculated from an alignment of 83 Saccharomycetales Rad5 sequences (top) or 395 fungal Rad5 sequences (bottom). Large letters above the zero line correspond to highly conserved residues in the alignment. The number of sequences with residues at this location is plotted underneath the sequence logo; note that the MIP box is present in almost all fungal Rad5 sequences aligned whereas the candidate SHIP box is present only in a small subset of the fungal Rad5 sequences corresponding to the Saccharomycetales (bottom). (C) Mapping of the predicted MIP and SHIP motifs (black spheres) onto the Alphafold2-predicted structure of S. cerevisiae Rad5 (Jumper et al., 2021; Varadi et al., 2022) reveals that these predicted motifs are in the unstructured N-terminus (black). (D) Yeast two-hybrid analysis shows that mutation of the predicted MIP box in the Rad5 bait vector retained the interaction with the Msh2 prey but resulted in a loss of interaction (indicated by no growth on selective -Leu -Trip -His medium) with the Mlh1 prey vector, whereas mutation of the predicted SHIP box in the Rad5 bait vector retained interaction with both the Msh2 and Mlh1 prey vectors.

Fig 3: Loss of SHPRH results in resistance to alkylating agents. (A) HeLa S3 and CRISPR MLH1 and MSH2 knockout cells were seeded into 6-well plates for 24 h followed by a 2-h pre-treatment with O6-benzylguanine and a 1-h treatment of MNNG with O6-benzylguanine and seeded at a low density into a 6-well plate for a clonogenic survival assay. Left panel is a representative of stained colonies. Right panel is cell viability with colony counting. Data is shown as the mean of N = 3 with 4 replicate wells each ± SEM. (B) HeLa S3 and CRISPR HLTF + SHPRH double knockout cells were treated the same as part A for the clonogenic survival assay. Data is shown as the mean of N = 3 with 4 replicate wells ± SEM. Survival is compared to HeLa MLH1 KO survival from part (A). (C) HeLa S3 and CRISPR HLTF knockout cells were treated the same as part A for the clonogenic survival assay. Data is shown as the mean of N = 3 with four replicate wells ± SEM. Survival is compared to HeLa MSH2 KO survival from part (A). (D) HeLa S3 and CRISPR SHPRH knockout cells were treated the same as part A for the clonogenic survival assay. Data is shown as the mean of N = 3 with 4 replicate wells ± SEM. Survival is compared to HeLa MLH1 KO survival from part (A). Statistical significance was determined by unpaired t-test *p < 0.05.

Fig 4: Rad5 has a predicted MIP and SHIP box and interacts with Mlh1 and Msh2. (A) The match score of 1,745 peptides from the nuclear S. cerevisiae proteome with a moderate or good motif matching to either the MIP or SHIP box motif as determined from bioinformatic analysis using a position-specific scoring matrix (PSSM) are plotted against their long-range disorder predicted by IUPRED (Dosztanyi et al., 2005). Rad5 was identified in analysis for both MIP and SHIP motifs. (B) Yeast two-hybrid analysis shows both Msh2 and Mlh1 prey constructs interact with Rad5 bait (growth on–Leu–Trp–His selective medium as well growth on the control–Leu–Trp medium). Exo1-C terminus bait shows positive interactions with Msh2 and Mlh1 prey as a positive control. Neither Rad5 or Exo1-C terminus bait constructs autoactivate in the presence of an empty prey vector.

Fig 5: Rad5 and human homologs interact with the MMR pathway. Saccharomyces cerevisiae helicase/E3 ligase Rad5 interacts with both key players in eukaryotic MMR, Msh2 and Mlh1. Rad5 has two human homologs, HLTF and SHPRH. Binding to the MMR pathway is conserved throughout evolution, but split between the two human homologs with HLTF binding MSH2 and SHPRH binding MLH1. SHPRH plays a role in apoptosis after alkylation damage as depletion of SHPRH results in mild resistance to MNNG. Created with BioRender.com.