Fig. 1.

Identification of MUS81 mutants that abolish interaction with SLX4. (A) MUS81 fragments used in the yeast two-hybrid assay. (B) Yeast were co-transformed with the bait and prey plasmid combinations indicated and plated on the media lacking leucine and tryptophan, with or without histidine (top and middle panels) or onto X-Gal (bottom panel). Y2H assays were performed with a GAL4 DNA binding domain (BD) fusion of MUS81 and activation domain (AD) fusion of SLX4 to detect interaction between these proteins. Cells grown on medium lacking LEU and TRP (to select for bait and prey plasmids) were replica-plated to medium lacking LEU, TRP and HIS to test for activation of the HIS3 reporter gene or tested for lacZ reporter gene activity. (C) Alignment of the N-terminus of MUS81 from different species. Residues were mutated in pairs of two to alanine (black lines). The residues that constitute the Helix-hairpin-Helix (HhH) are underlined in red. M.m., Mus musculus; H.s., Homo sapiens; X.t., Xenopus tropicalis; D.r., Danio rerio; D.m., Drosophila melanogaster; S.p., Saccharomyces pombe; S.c., Saccharomyces cerevisiae. (D) HEK293 cells were transiently transfected with plasmids expressing FLAG-EMPTY, FLAG-MUS81 wild-type (WT) or FLAG-MUS81 carrying double point mutants as shown in (C). Extracts were subjected to western blotting to test expression (upper panel) or immunoprecipitation with anti-FLAG antibodies (lower panel). (E) Immunoprecipitates from HEK293 cells co-transfected with plasmids expressing FLAG-MUS81 (WT), nuclease dead mutant FLAG-MUS81 D307A (D307A), FLAG-MUS81 loss-of-interaction mutants W24A L25A or L66A Q67A and FLAG-EME1 (EME1) were subjected to a nuclease assay with FITC-labeled 3′ flap substrate for time indicated, and the reaction products resolved by gel electrophoresis. FITC-labeled 3′ flap substrate and the cleaved duplex are shown on the right. Western blot confirming the immunoprecipitation with FLAG M2 beads is shown in the left panel.