Figure 7. Isolation and phenotypic analysis of rad50-46 intragenic suppressors.

A. Schematic illustration of the genetic screen to isolate intragenic suppressors of rad50-46 MMS sensitivity at 23°C. SacI digested plasmid Ycp50-RAD50 (from nucleotide 234-3225, corresponding to amino acid G78 – S1075) was gap repaired in rad50Δ cells with a rad50-46 PCR mutagenized repair template. Cells containing the gap repaired plasmid were plated on selective media containing 0.1% MMS and grown at 23°C. Plasmid were recovered from survivor colonies, re-transformed in rad50Δ cells, and suppression was confirmed on MMS plates. Sequenced rad50-46 suppressors are given. B. rad50-46 suppressors were re-assessed by spot test on plates with 0.1% MMS, either incubated at 23°C, 30°C or 37°C. Some of the rad50-46 suppressors were also assessed in context of the wild-type hook domain (bottom 4 strains). C. Mre11 complex integrity of rad50-46 suppressor mutants was assessed by co-immunoprecipitation and western blot with Rad50 or Mre11 antisera as indicated. D. Top: Sequence alignment of the central hook domain flanked by one coiled coil heptad repeat. The conserved zinc-cysteines are highlighted in green, rad50-46 suppressor mutation in yellow and partially conserved residues in grey. Arrows denote a structurally conserved inverted beta-sheet. pf, P. furiosus; sc, S. cerevisiae; sp, S. pombe; mm, M. musculus; hs, H. sapiens. Bottom: I-tasser structure prediction of the 41 amino acid (highlighted in blue) hook dimer peptide. The approximate position of the I680V, K700Q and L703F hook suppressor mutations are denoted. E. Circular dichroism spectra and UV spectroscopy of wild-type and rad50-46 hook peptides without and with hook suppressors. F. MMS spot test to assess suppression by N873I of other hook and coiled coil mutants. Plates were incubated either at 23°C, 30°C or 37°C.