Fig. 1: Identification of substitution mutations in the C-terminus of Rad50 that enhance Tel1 signaling.
From: Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms
A Working hypothesis. Sae2 targets Rad50 and regulates two MRX nuclease activities and Tel1 signaling through distinct mechanisms. B Rad50 protein domains and location of rad50 mutations. The rad50-C47 and rad50-C126 mutation are located at the C-terminus of Rad50. C Rescue of hydroxyurea sensitivity of mec1Δ cells by rad50 mutations. Serial dilutions of cultures were spotted on rich medium with or without hydroxyurea (HU). D Enhanced Rad53 phosphorylation in mec1Δ cells by rad50 mutations. Cells were treated with or without phleomycin and subjected to immunoblotting analysis with anti-Rad53 antibody. The wild-type (WT) strain, carrying both the wild-type MEC1 and wild-type RAD50 gene serves as a positive control. C47 or C126 denotes rad50-C47 or rad50-C126, respectively. The image is a representative of two independent experiments. E Alignment of the C-terminus of eukaryotic and prokaryotic Rad50. Eukaryotic Rad50 proteins possess conserved C-terminal tail regions. The dotted line (in red) corresponds to the circled area in (F). F The Rad50 coiled-coil region (in pink) folds back on itself and forms a rod-like structure, bringing the N- and C-terminal ATPase domains into the Rad50 head (in green). The diagram depicting C. thermophilum Rad50 was created by UCSF Chimera using the PDB data base (PDB ID: 5DAC). G Effect of the rad50-C47 or rad50-C126 mutation on Rad50 expression levels in vegetatively growing cells. Extracts were prepared from exponentially growing cells, transferred to a membrane, and stained with Ponceau S. Membranes were probed with anti-Rad50 antibodies. The strains used here carry RAD50, rad50Δ, rad50-C47 (C47) or rad50-C126 (C126). The image is a representative of three independent experiments. H Effect of the rad50-C47 or rad50-C126 mutation on Mre11-Rad50 interaction. Extracts were prepared from cells carrying HA-tagged MRE11 and untagged Rad50 variants, and subjected to immunoprecipitation (IP) with anti-Rad50 antibodies. The immunoprecipitates were further analyzed by immunoblotting with anti-HA or anti-Rad50 antibodies. The strains used here carry RAD50, rad50Δ, rad50-C47 (C47) or rad50-C126 (C126). The image is a representative of two independent experiments.
