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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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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).
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fig4: 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).

Mentions: We investigated the consequences of Mre11 inactivation on NHEJ by depleting Mre11 protein from cell-free extracts with a polyclonal serum specific for X. laevis Mre11 protein (anti-XMre11). We previously used these antibodies to demonstrate that genomic DNA accumulates DSBs during DNA replication in Mre11-depleted extracts (Costanzo et al., 2001). Moreover, Mre11 depletion dramatically impairs the ATM (Axatia telangiectasia mutated)-dependent response to DSBs (Costanzo et al., 2004). Importantly, we show that the former defect is rescued by the Mre11 hypomorphic ATLD3/4 allele, whereas ATM activation is not (Costanzo et al., 2004). This clearly demonstrates that Mre11-associated activity remains in ATLD cells. We used two types of cell-free extracts for our study and established that egg cytosol and membrane-free egg cytosol supported NHEJ with similar efficiency (see Figs. 2 and 4).


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

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

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171289&req=5

fig4: 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).
Mentions: We investigated the consequences of Mre11 inactivation on NHEJ by depleting Mre11 protein from cell-free extracts with a polyclonal serum specific for X. laevis Mre11 protein (anti-XMre11). We previously used these antibodies to demonstrate that genomic DNA accumulates DSBs during DNA replication in Mre11-depleted extracts (Costanzo et al., 2001). Moreover, Mre11 depletion dramatically impairs the ATM (Axatia telangiectasia mutated)-dependent response to DSBs (Costanzo et al., 2004). Importantly, we show that the former defect is rescued by the Mre11 hypomorphic ATLD3/4 allele, whereas ATM activation is not (Costanzo et al., 2004). This clearly demonstrates that Mre11-associated activity remains in ATLD cells. We used two types of cell-free extracts for our study and established that egg cytosol and membrane-free egg cytosol supported NHEJ with similar efficiency (see Figs. 2 and 4).

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