Fig 2: OTUB1 interacts with MSH2.A, Flag-MSH2 and HA-OTUB1 were cotransfected into HEK293T cells, and the transfected cells were then treated with 20 μM MG132 for 6 h before harvesting. Exogenous co-immunoprecipitation (Co-IP) was performed with Flag agarose beads or HA magnetic beads to detect interactions between Flag-MSH2 and HA-OTUB1. B, Co-IP was performed with anti-OTUB1 and anti-MSH2 antibodies to detect the endogenous interaction between MSH2 and OTUB1 in HEK293T cells. C, diagram showing OTUB1 fragments used in the GST pulldown assay. D, western blot of MSH2 (top) that was pulled down by purified GST-OTUB1 and its truncated fragments (bottom). E, diagram of MSH2 constructs used in the GST pulldown assay. F, purified GST-MSH2 polypeptides (bottom) were incubated with cell lysates of HeLa cells overexpressing HA-OTUB1, and HA was blotted as indicated (top).

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: MSH2/MSH6 Are Responsible for the Events in the Absence of rtel-1 and brc-1, but Not in him-6(A–E) Percentage of heterologous recombinant progeny in mismatch repair mutants (A), msh-2 combined with rtel-1, brc-1 or him-6 (B), msh-6 and pms-2 combined with rtel-1 (C), mlh-1 combined with rtel-1 (D), and mlh-1 combined with him-6 (E). n, number of worms scored. Statistics: χ2 tests or Fisher’s exact tests when n ≤ 1000.See also Table S1.