Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Cell Biol
2005 Dec 05;1715:765-71. doi: 10.1083/jcb.200506029.
Show Gene links
Show Anatomy links
Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.
Di Virgilio M
,
Gautier J
.
???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 gold–stained gel characterization of NHEJ products for substrate types nonmatching 3′-PSS (lanes 5–15) and blunt end + 3′-PSS (lanes 16–26). 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 + 5′-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 + 5′-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 3′-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).
Boulton,
Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing.
1998, Pubmed
Boulton,
Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing.
1998,
Pubmed
Chen,
Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.
2001,
Pubmed
Costanzo,
Mre11 assembles linear DNA fragments into DNA damage signaling complexes.
2004,
Pubmed
,
Xenbase
Costanzo,
Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication.
2001,
Pubmed
,
Xenbase
D'Amours,
The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.
2002,
Pubmed
Hollick,
2,6-disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-Dependent protein kinase (DNA-PK).
2003,
Pubmed
Huang,
Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction.
2002,
Pubmed
Khanna,
DNA double-strand breaks: signaling, repair and the cancer connection.
2001,
Pubmed
Labhart,
Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts.
1999,
Pubmed
,
Xenbase
Luo,
Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.
1999,
Pubmed
Manolis,
Novel functional requirements for non-homologous DNA end joining in Schizosaccharomyces pombe.
2001,
Pubmed
Moore,
Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.
1996,
Pubmed
Nussenzweig,
Requirement for Ku80 in growth and immunoglobulin V(D)J recombination.
1996,
Pubmed
Pfeiffer,
Joining of nonhomologous DNA double strand breaks in vitro.
1988,
Pubmed
,
Xenbase
Sandoval,
Joining of DNA ends bearing non-matching 3'-overhangs.
2002,
Pubmed
,
Xenbase
Stewart,
The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder.
1999,
Pubmed
Taccioli,
Impairment of V(D)J recombination in double-strand break repair mutants.
1993,
Pubmed
Theunissen,
Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice.
2003,
Pubmed
Thode,
A novel pathway of DNA end-to-end joining.
1990,
Pubmed
,
Xenbase
Varon,
Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome.
1998,
Pubmed
Wang,
Biochemical evidence for Ku-independent backup pathways of NHEJ.
2003,
Pubmed
Xiao,
Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.
1997,
Pubmed
Yamaguchi-Iwai,
Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells.
1999,
Pubmed
Zhong,
Deficient nonhomologous end-joining activity in cell-free extracts from Brca1-null fibroblasts.
2002,
Pubmed
Zhu,
Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.
2001,
Pubmed
