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???displayArticle.abstract??? Mre11-Rad50-Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.
Figure 1. Immunodepletion of Mre11 and Ku70 proteins from X. laevis egg extracts. (A) Untreated egg cytosol and mock-, Mre11-, or Ku70-depleted extracts were analyzed by Western blotting with either anti-XMre11 serum or antibodies against Ku70. (B) Untreated membrane-free egg cytosol and extracts treated with preimmune (mock depleted) or anti-XMre11 serum (Mre11 depleted) were analyzed by Western blotting with anti-XMre11 serum. (C) Mre11 and Ku70 double-depleted egg cytosol (Mre11 and Ku70 depleted) were analyzed with either anti-XMre11 serum or antibodies against Ku70. (D) Untreated egg cytosol, Mre11-, or Mre11 and Ku70 double-depleted extracts were incubated with streptavidin beads coated with biotinylated, linear double-strand DNA, and the fraction of proteins bound to DNA was analyzed by Western blotting with anti-XMre11 serum.
Figure 2. Mre11 is not required for efficient NHEJ. (A) Representative SYBR goldâstained gel characterization of NHEJ products for substrate types nonmatching 3â²-PSS (lanes 5â15) and blunt end + 3â²-PSS (lanes 16â26). Lanes 5 and 16 show 10 ng of input SacI-KpnI and SacI-SmaI substrates, respectively. The input in lane 2 corresponds to 10 ng of undigested pBS KS II. mt, mitochondrial DNA from X. laevis extract; M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle. (B) The intensity of the bands corresponding to NHEJ products were quantified using the FluorImager system for each NHEJ reaction in Ku70- and Mre11-depleted extracts and were expressed as percent repair efficiency (see Results). The labeling on the x axis refers to the NHEJ substrate type as indicated in Table SI (available at http://www.jcb.org/cgi/content/full/jcb.200506029/DC1).
Figure 3. Mre11 depletion does not affect NHEJ-mediated generation of multimers. (A) Southern blot analysis of NHEJ products generated by incubation of blunt end + 5â²-PSS substrate in the indicated depleted egg cytosol extracts at a substrate input concentration of 0.1 (left), 1 (middle), or 10 (right) ng/μl. Data in the left panel are derived from two segments of the same gel (as indicated by the dividing line). (B) Blunt end + 5â²-PSS substrate was incubated at 1 ng/μl in untreated egg cytosol that had been supplemented with the recombinant MRN complex, and NHEJ products were analyzed by Southern blot hybridization. M, higher multimer forms; LD, linear dimer; LM, linear monomer (substrate); CC, closed circle; OC, open circle.
Figure 4. NHEJ kinetics is not affected by Mre11 depletion. Nonmatching 3â²-PSS substrate SacI-KpnI was incubated in mock- and Mre11-depleted membrane-free cytosol, and samples were taken at the indicated time points. DNA was recovered and analyzed by (A) colony formation assay and (B) Southern blot hybridization (grouping of different segments of the same gel is indicated by dividing lines).
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