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Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.

Di Virgilio M, Gautier J - J. Cell Biol. (2005)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, NY 10032, USA.

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.

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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.
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fig1: 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.

Mentions: Mre11 protein was quantitatively removed by two rounds of immunodepletion and was no longer detectable by Western blotting (Fig. 1 A, lanes 2–5; and B). Mre11 depletion did not affect Ku70 protein level (Fig. 1 A, lanes 2–5). Conversely, Mre11 protein levels were not reduced by Ku70 depletion (Fig. 1 A, lanes 6–9). This demonstrates that there is no significant interaction between the two proteins in solution, and it allows for the independent analysis of Mre11- and Ku70-depleted extracts. Moreover, both Mre11 and Ku70 proteins can be depleted simultaneously from egg extracts (Fig. 1 C).


Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.

Di Virgilio M, Gautier J - J. Cell Biol. (2005)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171289&req=5

fig1: 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.
Mentions: Mre11 protein was quantitatively removed by two rounds of immunodepletion and was no longer detectable by Western blotting (Fig. 1 A, lanes 2–5; and B). Mre11 depletion did not affect Ku70 protein level (Fig. 1 A, lanes 2–5). Conversely, Mre11 protein levels were not reduced by Ku70 depletion (Fig. 1 A, lanes 6–9). This demonstrates that there is no significant interaction between the two proteins in solution, and it allows for the independent analysis of Mre11- and Ku70-depleted extracts. Moreover, both Mre11 and Ku70 proteins can be depleted simultaneously from egg extracts (Fig. 1 C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, NY 10032, USA.

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.

Show MeSH