Fig. 3: Mutagenesis of conserved Rad50 interface regulates MR oligomerization.

a Crystal structure of two C. thermophilum (Ct) Rad50 dimers bound to one oligonucleotide molecule each (PDB: 5DAC⁴⁸, 10.2210/pdb5dac/pdb) used to generate a plausible interaction model as guide for validation by mutagenesis. Possible interactions of adjacent Rad50 dimers on DNA via a small beta-sheet (β) and its connecting loop (L) protruding from the Rad50 head domain (rectangle), especially if bound DNA was continuous. Zoom-inset shows red/blue residues mutated in the S. cerevisiae (Sc) Mre11-Rad50^ho/lo variants. Gray dotted line: salt bridge between E125 and R126 residues of adjacent CtRad50 dimers (spacing 3.8 Å within crystal). Red dotted line: repulsive E125 residues of adjacent CtRad50 dimers are spaced by 4.8 Å within the crystal, but could come closer on continuous DNA and in solution. b Sequence alignment of the Ct, Sc, and H. sapiens Rad50 beta-sheet motifs. Color code as in a. * indicates germline point mutations listed on ClinVar/MedGen for cancer-predisposed humans. Cartoons show multiple point mutations introduced in ScRad50 to disrupt electrostatic (Rad50^ho) or hydrophobic (Rad50^lo) interactions (Supplementary Fig. 5a). c Thioflavin T (ThT) fluorescence of ScMR^wt indicates beta-sheet rich higher-order structures (Supplementary Fig. 5e). Mean ± SEM, n = 4 independent experiments, unpaired two-tailed t-test, a.u., arbitrary units. d (i) Representative TEM of ScMR^wt/ho/lo as in Fig. 1a–v. (ii) Quantification of mean head domain diameters ± SEM of ScMR^wt/ho/lo oligomers from (i) along their longest axis. n_MR-wt = 30 molecules, n_MR-ho = 72, and n_MR-lo = 46. Gray area indicates diameter range of ~8–14 nm measured for ScMR^wt dimers. Scale bar: 100 nm. e Molecular mass distributions from mass photometry show better ScMR^ho binding to longer oligonucleotides (100 nM base pairs). Cartoons illustrate species in peaks. Measured molecular weight (MW, kDa) ± SD in gray. Zoom-insets show MW range where a peak of two MR dimers bound to one DNA molecule would be expected and was fitted where possible (Supplementary Fig. 3b). n = 3 independent experiments. f Quantification of ScMR^wt/ho pelleting assays at 4 or 42 °C (see Supplementary Fig. 5c). Sup. supernatant. Higher temperature promotes oligomerization. Mean ± SEM, n = 3, unpaired two-tailed t-tests.