Fig 2: The MSH2 binding domain of SLX4 is a SHIP box that inhibits mismatch repair and antagonizes EXO1–MSH2 interaction. (A) Weblogo representation of multiple sequence alignments of SHIP boxes of Exo1 and Fun30 from fungal species in the Saccharomycotina (46,82). (B) HeLa FITo cells were transfected with expression vectors coding for GFP, FLAG-SLX4, GFP-MSH2 WT and/or GFP-MSH2M453I, as indicated, before GFP pull-down and western blotting with the indicated antibodies. The asterisk represents the remaining signal for EXO1 when blotting for MSH3 after prior blotting for EXO1. (C) Peptide pull-down using biotinylated SLX4 peptides that contain a WT or mutated SHIP box, immobilized on Streptavidin-coated beads and incubated with HeLa nuclear extracts. Residues selected for mutagenesis are shown in bold in the WT sequence. (D) In vitro mismatch repair assay using a plasmid containing a G/T mismatch and a 5′ nick incubated with nuclear extracts (NE) from HeLa FITo cells as described in Material and Methods. Where indicated, WT or mutant SHIP peptides (80.5 μM) were added to the reaction. As a negative control, the NE was inactivated at 95°C before the reaction. DNA was purified and digested with ApalI and PvuII. Repair of the mismatch restores the PvuII site and produces two bands of 1.55 and 1.03 kb on an agarose gel. The percentage of repair is indicated.