(A) The amino acid sequences of Mre11 proteins from Homo sapiens (ACcession number: P49959), Mus musculus (AC: Q61216), Gallus gallus (AC: Q9IAM7), and Xenopus laevis (AC: Q9W6K1) were aligned using the CLUSTAL W (1.83) multiple sequence alignment program. SQ/TQ motifs are highlighted in bold. The amino acid positions of the Ser/Thr residues in the eight SQ/TQ motifs of the human protein are indicated. ND: Nuclease domain; DBD-1/2: DNA-binding domain 1/2. (B) Purified MRN-WT, MRN-SA, and MRN-SD complexes were immunoprecipitated with preimmune (Pre) or anti-XMre11 (αMre11) serum, and the immunoprecipitates were immunoblotted with anti-hRad50 serum (upper panel), anti-hNbs1 serum (middle panel), and anti-XMre11 serum (lower panel). The inputs shown are equivalent to 15% of the purified MRN complex used in the co-immunoprecipitation reactions. (C) Mre11-depleted membrane-free extracts supplemented with recombinant MRN-WT or MRN-SA complex were incubated for 15 min in the presence or absence of DSBs (4.34 × 10¹⁰ ends/μl), okadaic acid (OA; 4 μM), and KU55933 (10 or 100 μM) or caffeine (10 mM), and analysed by Western blotting with anti-XMre11 serum.

(A) Mock- and Mre11-depleted membrane-free egg extracts supplemented with MRN-WT, -SA, or -SD (500 nM) were incubated with biotinylated 1 Kb linear double-stranded DNA (B-1 Kb DNA) bound to streptavidin beads and free radioactive non-biotinylated 1 Kb DNA. Streptavidin-immobilized DNA fractions were isolated, and the associated radioactivity (cpm) was measured. Values are expressed as percentages relative to B-1 Kb DNA-supplemented Mock-depleted sample, which is set to 100%, and error bars represent standard deviations from three independent experiments. (B) Mock- and Mre11-depleted membrane-free egg extracts supplemented with MRN-WT, MRN-SA, or MRN-SD (500 nM) were incubated with B-1 Kb DNA (1.1 × 10¹¹ ends/μl) bound to streptavidin beads for 10 min at 22°C. The DNA-bound fractions were separated from the supernatants and analyzed by Western blotting with antibodies against γ-H2AX (upper panel) and MCM6 (lower panel; loading control).

Interphase egg cytosol (A) and membrane-free egg cytosol (B) extracts were incubated for 15 min with DSBs-containing DNA (DSBs) at the indicated concentrations in the absence or presence of 4 μM of okadaic acid (OA), and analyzed by Western blotting with anti-XMre11 serum. (C) Membrane-free egg cytosol was incubated for the indicated times with DSBs (4.34 × 10¹⁰ ends/μl) in the absence or presence of 4 μM of okadaic acid (OA), and analyzed by Western blotting with anti-XMre11 serum. (D) Untreated interphase egg cytosol, mock-, and Mre11-depleted extracts supplemented with recombinant MRN complex were incubated with DSBs (4.34 × 10¹¹ ends/μl) in the presence or absence of tautomycin (4 μM), and analyzed by Western blotting with anti-XMre11 serum. (E) Mock- and Mre11-depleted membrane-free extracts supplemented with purified MRN complex were incubated in the presence or absence of DSBs (4.34 × 10¹⁰ ends/μl) and OA (4 μM), treated with Lambda Protein Phosphatase (λ-PPase) (lanes 5 and 9), and analyzed by Western blotting with anti-XMre11 serum.

(A) Untreated membrane-free egg extracts were incubated with biotinylated 150 bp linear double-stranded DNA (B-150 bp DNA) bound to streptavidin beads for the indicated times in the absence (lanes 1-5) or presence (lanes 6-9) of 4 μM okadaic acid (OA). The DNA-bound fractions were pulled down, electrophoresed on NuPAGE® Novex 3-8% Tris-Acetate gel and analyzed by Western blot with anti-XATM serum (upper panel) or anti-XMre11 serum (lower panel). (B) Mre11-depleted membrane-free egg extracts supplemented with MRN-WT, -SA, or -SD recombinant complex were incubated for 30 min with B-150 bp DNA bound to streptavidin beads. The resulting supernatants were separated from the DNA-bound fractions, and both fractions were analyzed by Western blotting with antibodies against the indicated proteins. (C) Mre11-depleted membrane-free egg cytosol supplemented with MRN-WT or MRN-SA complex was incubated with B-150 bp DNA bound to streptavidin beads for the indicated time points. The DNA-bound fractions were separated from the supernatants, and analyzed by Western blotting with anti-XMre11 serum (upper panel) and anti-hNbs1 serum (lower panel). (D) Mre11-depleted interphase egg cytosols supplemented with MRN-WT, MRN-SA, or MRN-SD complex were incubated for 1 h with demembranated sperm nuclei (10,000 nuclei/μl), and the chromatin fractions were isolated and analyzed by Western blotting with anti-XATM serum (upper panel) and anti-XMre11 serum (lower panel). (E) Mre11-depleted membrane-free egg extracts supplemented with either MRN-WT or MRN-SA complex were incubated for 5 min at 22 °C with biotinylated 150 bp linear double-stranded DNA (B-150 bp DNA) bound to streptavidin beads. The DNA-bound fractions were separated from the supernatants and used as a substrate for an in vitro kinase reaction in the presence or absence of purified monomeric ATM kinase (mATM) and dATP. DNA-bound proteins were then separated on SDS-PAGE, and analyzed by Western blotting with anti-XMre11 serum (upper panel), anti-hNbs1 serum (middle panel), and anti-hRad50 serum (lower panel).