Fig. 4: MR oligomerization influences DNA binding behavior and endonuclease activity.

a Quantification of EMSAs of MR^wt/ho/lo binding to 100 bp-long DNA as in Supplementary Figs. 2a and 5f. Mean ± SEM, n = 3 independent experiments, sigmoidal fit. b TEM of MR^ho/lo binding to plasmid DNA. Images representative of n = 3 experiments. MR^lo binds dispersedly on DNA without oligomerization, while MR^ho dimers oligomerize on DNA to a denser “worm”-like structure compared to the “pearls-on-a-string” assembly of MR^wt (Fig. 1b-ii) at similar conditions (see “Methods”). c TEM of Mre11^wt and Rad50^wt/ho/lo binding to plasmid DNA. n = 3. In contrast to purified Mre11^wt, Rad50^wt forms “pearls-on-a-string”-like structures on linear plasmid DNA. Rad50^lo fails to oligomerize, thus binds dispersedly on DNA, while Rad50^ho clusters into large oligomers on DNA, similarly to MR^ho. d, e Endonuclease assays of MR^wt/ho/lo-pSae2 (d, Supplementary Fig. 6a) with quantification (e). Images representative of n = 3 experiments. Mean ± SEM, unpaired two-tailed t-tests. Red star marks the position of the radio-label. f, g Representative endonuclease activity kinetics of MR^wt/ho-pSae2 (f) with quantification (g). Mean ± SEM, n = 3 independent experiments. Red star marks the position of the radio-label. h Representative resection assay of MR^ho with probe (green box in cartoon) binding to the 3′-overhang produced. n = 2. Note that the large MR^ho oligomers in Fig. 4b are active in resection. i Interaction assay of MR^wt/ho/lo with the C-terminal part of pSae2 (pSae2 \(\triangle\)N169). Ponceau and anti-MBP western blot indicate elution of MBP-tagged pSae2 \(\triangle\)N169. The anti-Rad50 western blot shows interaction of pSae2 \(\triangle\)N169 with Rad50 in MR^wt/ho/lo. One out of two independent experiments is shown. Scale bars: 100 nm (b, c).