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. Author manuscript; available in PMC: 2010 Nov 2.
Published in final edited form as: DNA Repair (Amst). 2009 Aug 25;8(11):1311–1320. doi: 10.1016/j.dnarep.2009.07.006

Figure 3. Phosphorylation of Mre11 SQ/TQ motifs modulates MRN complex association with DNA.

Figure 3

(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).