Figure 3.

Mirin inhibits the nuclease activity of Mre11. (a) Mirin does not trigger MRN complex dissociation in extracts. Extracts were incubated with FLAG-tagged MRN complex and then incubated with DMSO (−) or mirin. Recombinant MRN was isolated, and supernatants or FLAG-resin were probed using antibodies against FLAG, Mre11 or Rad50. X. laevis Rad50 protein is not recognized by the Rad50 antibody. (b) Mirin does not dissociate endogenous Mre11-associated complexes. Extracts were treated with DMSO or 100 μM mirin and fractionated on a Superose 6 gel-filtration column. Fractions were probed by western blot for Mre11. Molecular weight markers are indicated on top. (c) Mirin does not inhibit ATM and Mre11 binding to DNA. Extracts were treated with DMSO (−) or with mirin before incubation with DSB-containing DNA (1.2 × 10¹¹ ends μl⁻¹). Following DNA pull-down, phosphorylation of ATM Ser1981 (upper panel), ATM (middle panel) and Mre11 (lower panel) were monitored by western blotting in soluble fractions and DNA fractions. (d) Mirin does not inhibit MRN-associated DNA tethering activity. Mock-treated (dark gray) or Mre11-depleted (light gray) extracts were incubated with DMSO (−) or mirin at the indicated concentrations. Extracts were then incubated with streptavidin-bound DNA and free radioactive DNA. DNA-tethering activity was assayed by measuring the radioactivity associated with streptavidin-bound DNA. Each bar represents an average of six independent experiments with s.d. shown. Values marked with asterisks are significantly different (P = 0.001). (e) Mirin inhibits the nuclease activity of MRN. Purified human MRN was incubated with a 5′-labeled double-strand oligonucleotide in the presence of DMSO or mirin, as indicated. Digested DNA products were analyzed by denaturing PAGE followed by autoradiography